Synthesis of Florbetapir aza-analogues using chemistry of pyridinium N -aminides

Neuroimaging of β -amyloid (A β ) plaques in brain, employing Positron Emission Tomography (PET) has enabled early diagnosis of Alzheimer’s Disease and, although different 18 F radiolabeled markers as Florbetapir and Florbetaben are already in the market, new molecules with better affinity and selectivity to A β plaques should be explored. In this article, two aza-analogues of Florbetapir have been synthesized from Pyridinium N -aminides. The new aza-analogues were prepared following straightforward synthetic routes under mild conditions. Although the products have been obtained using stable 19 F, the methods are compatible with the future use of 18 F, to generate products to be tested in the development of new PET reagents.


Introduction
Alzheimer's disease (AD) is the most common dementia among the population, with a marked increase in the number of cases being expected in the future, due to the increase in life expectancy.There is currently no cure for AD, it is an irreversible illness and current therapies only slow its process, therefore early diagnosis is essential for an effective treatment. 1Unfortunately, the diagnosis of a possible case of AD, based exclusively on the patient's medical history, can only be made once the cognitive decline is severe, as the initial symptoms of the illness are difficult to differentiate from other dementias.][3] The pathogenesis of AD is complex and characterised by an abnormal β-amyloid metabolism, hyperphosphorylation of tau protein and other pathological processes in the central nervous system. 4,5ccording to the amyloid hypothesis, there is a presymptomatic state of AD which begins with an imbalance between the production and clearance of β-amyloid protein (Aβ) in the brain, thus resulting in the accumulation and aggregation of Aβ, in the form of plaques, in the grey matter that triggers the neurodegenerative cascade seen in AD. [5][6][7][8][9][10][11] These plaques are absent in other types of dementias, and in the diagnosis of AD it is estimated that their progressive accumulation begins between 20 and 30 years before the symptomatic phase, thus meaning that Aβ plaques are excellent biomarkers for an early diagnosis. 7[14]  Although this first generation of PET markers is still used, there is a growing interest in the development of new radiotracers whose properties may allow the detection of Aβ plaques with better selectivity and sensitivity, with styrylbenzene derivatives, [15][16][17][18] benzoheterocycles, 6,[19][20][21][22][23][24] and metallic complexes 20,25,26 being important in this regard.One of the strategies adopted to enhance the selective delivery of the PET marker to the grey matter involves the design of less lipophilic molecules. 3,6Given this, we planned on the structure of Florbetapir, to prepare aza-analogues 1, with azo groups similar to those present in Congo red and Chrisamine G, for evaluation as PET radiotracers.Calculated log P (ClogP) values have been taken as an orientation of the relative lipophilicity (Florbetapir ClogP 3.11), both the presence of an azo group and a 2-aminopyridine moiety would slightly decrease the lipophilicity of these new potential radioligands and favour the affinity of 1 with the Aβ-plaques (Figure 2).The synthesis of Florbetapir aza-analogues 1 was planned using acetamides 4 as key intermediates.These compounds were obtained, according to a previously published methodology which involves the regioselective C-N coupling of pyridinium aminide 2 27 with a diazonium salt, and transformation of the resulting ylide 3 into 4, by N-exo alkylation and subsequent N-N cleavage under reducing conditions (Scheme 1). 28Two pathways were planned in order to obtain the final products 1 from 4. Thus, whereas in route A aniline-type amino group is protected until the fluorine atom has been introduced into the molecule, in order to avoid possible side reactions, thus requiring a final step of hydrolysis of the amide, in the route B fluorination is the last step in the synthesis, thereby avoiding the need of an additional deprotection of the amino group, after the fluorine label has been introduced into the molecule (Scheme 1).Both schemes are suitable to be used in the preparation of PET reagents.Scheme 1. Synthetic schemes of products 1.

Synthesis of acetamides 4
As already noted, synthesis of acetamides 4 was planned employing the pyridinium N-(pyridin-2-yl)aminide 2 as starting material, 27 in a three step procedure that starts with the preparation of arylazo derivative 3 (Scheme 2). 28Addition of the freshly prepared diazonium salt 5 to a solution of aminide 2, containing the base at 0 °C yielded the desired aminide 3 in 72% yield, together with its minor isomer 6 (11%).Regioselectivity of the process is increased by reducing the temperature to -20 °C, and then 3 was the only product isolated (95% yield, Scheme 2).Scheme 2. Synthesis of aminide 3.
Alkylation of aminide 3 proved to be challenging because of the relatively low reactivity of both alkyl bromides 7. 28 However, reaction was achieved by MW irradiation of the mixture in the absence of solvent, obtaining a mixture of salts 8 and 9, produced by alkylation at each exocyclic and endocyclic nitrogen of the aminide (Scheme 3).Both temperature and reaction time were explored in order to improve regioselectivity and the best results are indicated (see Suppl.Table 1).As attempts to purify the reaction mixture proved unsuccessful, conversions and exo/endo ratios were deduced by 1 H NMR spectroscopy, with salt 8 being the major product in all cases.Scheme 3. Synthesis of acetamides 4 from aminide 3.
The mixtures 8/9, in which conversion was complete, were treated with the reducing system HCOOH/Et3N in the presence of Pt/C as catalyst, to yield aminopyridines 4 (Scheme 3) as only the N-N bond easily cleaves under reduction.Product 4a was obtained using a 5% Pt/C catalyst, 28 (28% yield), while 4b required the use of 1% Pt/C, (21% yield).In both cases, compound 9 resulted stable in the reduction step.

Route A
This route began with elimination of the protecting benzyl group from acetamides 4, by treatment with 48% hydrobromic acid (Scheme 4).Compounds 10 were obtained in good yields after reaction for 16 h at room temperature (Table 1).Decomposition was observed when higher temperatures were employed, in an attempt to accelerate the process.However, the reaction time was drastically reduced when the process was performed at 40 °C under microwave irradiation, giving similar slightly lower yields of 10 in 30 minutes (Table 1).The hydroxy group of both compounds was activated by reaction with tosyl chloride in the presence of triethylamine and DMAP, leading to tosylates 11 after reaction at room temperature for 2 h (Scheme 4).  ) in acetonitrile, at 80 °C for 10 minutes (Scheme 5).However, while derivative 12b was obtained in 63% yield, under the same reaction conditions, tosylate 11a (n = 2) was transformed into the morpholine derivative 13, due to an intramolecular nucleophilic substitution (Scheme 5).
Given this result, we became interested in finding conditions that favour the fluorination of 11a over cyclization.In general, fluoride ions, either associated to kryptofix [2.2.2] or not, are known to act as a base.Experiments were performed with longer times, and when the same mixture was left to react for longer, up to 21 h, again tosylate 11a was transformed into the morpholine derivative 13, but the desired fluorinated product 12a was detected (~3-5%) for the first time.Also, 11a was treated with KF in the absence of cryptand, at 80 °C for 10 minutes, with only starting material being recovered.Alternatively, the treatment of 11a with an excess of TBAF (3 equiv.) in tBuOH/CH3CN, at 80 °C for 5 h, yielded the fluorinated compound 12a as the major product (49%), together with 19% of 13 (Scheme 5).The reaction time was reduced to 20 min when the same reaction was carried out at 120 °C using isoamyl alcohol/CH3CN as solvents, and 12a (69%) and 13 (14%) were obtained (Scheme 5).

Scheme 5. Fluorination of tosylates 11.
Having obtained the fluoro derivatives 12, to convert them in compounds 1, it was necessary the final acetamide deprotection.Deacylation under acid conditions was discarded because azo-derivatives could decompose in acid media.Alternatively, deprotection in basic media requires longer reaction times.In our case, refluxing acetamide 12b with 2 M NaOH in methanol for 5 h, gave 1b (79%) along with unreacted starting material (Scheme 6).However, the use of microwaves allowed deprotection under overheating conditions, thus showing that the reaction was significantly accelerated, giving 1 in 15 minutes, with excellent yields either for 1a or for 1b (Scheme 6 and Table 2).Scheme 6. Synthesis of compounds 1.

Route B
Although "cold" Florbetapir aza-analogues 1 could be obtained following route A, in the synthesis of any PET radiotracers, to make additional reaction steps after fluorination is undesirable because of the short half-life of the 18 F isotope.For this reason, and in order to avoid additional steps after fluorination, the alternative synthetic pathway B was tested.Synthesis of alcohols 14 was achieved by treating compounds 10 (route A) with 2 M NaOH in methanol under microwave irradiation, giving the products 14 in excellent yields (Scheme 7).Alternatively, compounds 14 were also obtained from 4 via amines 15 (Scheme 7).In both cases, when the amide deacetylation was performed either before or after removal of the benzyl group, both schemes took place satisfactorily, thus allowing us to conclude that the order of the two deprotection steps has little effect on the overall synthetic yield.However, from our point of view, it is more convenient to deprotect the hydroxy group first because the synthesis of 10 is common to route A and the yields obtained are slightly higher.

Scheme 7. Synthesis of alcohols 14.
Initial attempts to tosylate 14b using tosyl chloride, Et3N and DMAP in dichloromethane yielded a mixture of compounds 16, 17 and 18 along with unreacted starting material, thus indicating a non-selective reaction (Scheme 8).Alternatively, activation of products 14 resulted in better yields when using NaOH in aqueous media, which allowed the required tosylation on the hydroxy group (Scheme 8).Although the yields obtained were not as good as expected, the advantage of this process is that unreacted 14 was almost completely recovered and the formation of side products 17 and 18 was reduced to traces.

Scheme 8. Tosylation of compounds 14.
As in route A, fluorination of tosylate 16b, which contains the longer polyethoxy chain, was achieved using KF and kryptofix[2.2.2] in only a few minutes, whereas the same treatment of compound 16a yielded the morpholine derivative 19 (Scheme 9).Although the undesirable cyclization process was not completely avoided, rapid fluorination of 16a was achieved using TBAF in isoamyl alcohol/acetonitrile, thus giving 1a in 55% yield (Scheme 9).Scheme 9. Fluorination of tosylates 16.

Conclusions
Two Florbetapir aza-analogues, bearing diazo groups have been synthesised following simple and straightforward processes, starting from pyridinium N-aminides, by reaction with diazonium salts.With the bottleneck of the lack of selectivity of the alkylation of the exocyclic nitrogen of the 2-aminopyridine moiety, two routes have been studied as different approaches to generate Florbetapir analogues labelled with F. From both approaches studied, the route B, with fluorination in the last step, and the compound 1b, which prevents the intramolecular cyclisation, seems to be the best choice.The synthesis described would be tested in a program to develop new PET-radiotracers for brain imaging of β-amyloid plaques.In addition, synthetic efforts have resulted in the optimization of some common reactions in organic synthesis, as the microwave-assisted selective deprotection of alcohols and amines, and the fluorination of alkyl chains where a cyclization process is a competing reaction.

Experimental Section
General.ClogP values were estimated using the MarvinSketch 18.2 program.Melting points were determined in open capillary tubes using a Stuart Scientific SMP3 melting point apparatus.IR spectra were obtained using a Perkin-Elmer FTIR spectrophotometer. 1 H and 13 C NMR spectra were recorded using Varian Unity 300/500 MHz or Varian Mercury VX-300 systems at room temperature.Chemical shifts are given in ppm (δ) downfield from TMS.Chemical shifts in 19 F NMR spectra are reported in ppm (δ) with PhCF3 as internal standard (PhCF3: -63.46 ppm).Coupling constants (J) are in Hertz (Hz) and signals are described as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; app, apparent; br, broad.The numbering employed in NMR analysis is described in the Supporting Information.Low resolution mass spectra (MS) were recorded using a Thermo Scientific ITQ900 system with Electronic Impact (EI), and high-resolution analysis (TOF) was performed using an Agilent 6210 timeof-flight LCMS system with Electro Spray Ionization (ESI).All reagents were obtained from commercial sources and were used without further purification.TLC analyses were performed on silica gel (DCFertigfolien ALUGRAM Xtra Sil G/UV254, Macherey-Nagel) and spots were visualised under UV light.Column chromatography was carried out on silica gel 60 (40-63 mm, Silicycle) columns using the eluent reported in each case.Microwave experiments were performed using a Biotage Initiator and sealed 2 or 5 mL Biotage vials.This is a single-mode operating system, working at 2.45 GHz, with a programmable power level from 0-400 W. Stirring was performed at 400 rpm with the magnetic stirrer included in the apparatus and Temperature was measured using an external surface sensor.