Heterocycles for Alzheimer Disease: 4- and 5-Substituted Benzothiophenes as Starting Scaffold in the Construction of Potential New Inhibitors of BACE 1

Copyright: © 2016 Funicello M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Heterocycles for Alzheimer Disease: 4and 5-Substituted Benzothiophenes as Starting Scaffold in the Construction of Potential New Inhibitors of BACE 1


Introduction
Heterocycles are among the earliest organic compounds to be purified and recognized as discrete substances, although their structures remained unknown for a long time. The science of chemistry had a rapid progress since the first half of the nineteenth century [1] and the interest in heterocycles has increased over the years and continues to captivate many chemists worldwide. In particular, medicinal chemistry is intimately associated with heterocyclic compounds: in fact most of all known drugs contain heterocyclic frameworks [2].
In our long research on heterocyclic synthesis and application [28], we focused, in particular, on substituted benzothiophenes, namely 4and 5-derivatives, and here we will discuss their preparation and their use as starting scaffold for the synthesis of more complex heterocyclic structure such as functionalized benzothienopyridines.
Alzheimer Disease is a neurological pathology that in the last ten years is increasingly widespread and still can not cure. Moreover it's well known that BACE1 plays a key role in the development of Alzheimer's disease and its inhibition is a therapeutic target [29][30][31]. Hence we will report the synthesis and activity of benzothieno[b]pyridines, small tricyclic molecules just known for many other biological activities.

Experimental General
Column chromatography was carried out on Merck silica gel (0.063-0.200 mm particle size) by progressive elution with petroleum ether/ethyl acetate or petroleum ether/diethyl ether mixtures. 1 H-Abstract 4-and 5-substituted benzothiophenes were synthesized as starting scaffold in the construction of benzothieno [b] pyridines. Such structures were designed as potential new inhibitors of BACE 1. Preliminary promising biological data were reported. These molecules represent a starting point for improved chemicals in Alzheimer's disease treatment. Obtained preliminary results encourage research advance and further developments. and 13 C-NMR spectra were normally recorded for CDCl 3 solutions on a Bruker AM 300 MHz or on Varian INOVA 400 and 500 MHz instruments. IR spectra were registered on a JASCO FT/IR 460 Plus. Mass spectra were obtained with a Hewlett-Packard 5971 mass-selective detector on a Hewlett-Packard GC/MS 6890-5973 system. Dichloromethane was dried with anhydrous CaCl 2 ; THF was dried using sodium/benzophenone. Dry dimethylformamide was commercially available.

5-Br-4-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene (2)
In a two-neck flask, 4-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene (1) (504 mg, 3.31 mmol) was dissolved in 40 mL of anhydrous CH 2 Cl 2 . Under argon atmosphere, 2-pyrrolidinone hydrotribromide (1.80 g, 3.64 mmol) was slowly added and the mixture was stirred at room temperature for 19 hours. Then the mixture was heated (50°C) for 2 hours. The quench is performed by addition of saturated solution of Na 2 S 2 O 3 , extraction and washing with HCl 10%. The organic phase was dried over Na 2 SO 4 and then concentrated under reduced pressure. The product (2) was obtained as yellow solid. Compounds 40 and 41 were obtained as byproduct in the Staudinger reaction: they are known compounds and here we report only NMR and MS charactherization.

Results and Discussion
A large number of methods to synthesize heterocycles containing benzothiophene ring have been reported in recent years [9,[33][34][35], most of which involve the cyclization of benzenethiol derivatives. However, facile and versatile methods to access multi-substituted benzothiophenes are still limited. Furthermore, catalytic cyclization approaches using transition metals for the construction of the benzothiophene skeleton, which would provide a more efficient and practical route, are extremely rare in literature, presumably due to catalyst poisoning by sulfur; only few recent reports are known [36][37][38][39][40] and the described methodology is not applicable to the synthesis of all the substituted benzothiophenes.

Synthesis of 4-substituted benzo[b]thiophenes
Different ways are known for the preparation of 4-substituted benzothiophenes, but, generally, the retrosynthetic approach to the synthesis can be easily showed as in the next Scheme 1.
As regards 4-hydroxybenzothiophene (3) we initially employed the classical synthesis in two steps (bromination followed by elimination of bromide and aromatization) shown in Scheme 2. During this reaction we observed the formation of the dibromide byproduct (2a in Figure 1) which, in the next step, results in the formation of 5-Br-4-hydroxybenzothiophene (3a in Figure 1) hardly separable from the desired product (3) by chromatography with consequently low yield (45%).  The importance of 4-hydroxybenzothiophene as key intermediate for the synthesis of the other 4-substituted benzothiophenes had required further investigation in order to improve the yield and to find mild conditions avoiding the use of CCl 4 (a known carcinogen) and of molecular bromine because of its toxicity and difficulties associated with its manipulation (very corrosive liquid, highly toxic fumes).
So it was developed a new procedure (Scheme 3) which uses PHT (pyrrolidone hydrotribromide) as a brominating agent, according to a similar procedure reported in the literature [41].
The bromination was conducted in different solvents and experimental conditions in order to identify the optimal procedure. The reactions were followed by GC/MS in Table 1 was reported the percentage of conversion relative to the two brominated species. As shown in entry 6, reported conditions allow obtaining only the desired product in 86% of conversion. Applying the new methodology, it was possible to obtain an improvement of the overall yield of 4-hydroxybenzothiophene from 45% to 65% (Scheme 4).
Furthermore, we will emphasize how the new procedure provides very mild bromination conditions compared to the previous: the brominating agent (PHT) is a non-toxic non-corrosive solid and the reaction is conducted at room temperature and only for 2 hours under reflux of CH 2 Cl 2 (40°C).   could be introduced both during the synthetic route or subsequently by modifying or using those already present (i.e., coupling with amino acids or similar).
The criteria for the choice of the synthetic strategy were the necessity of particular functional groups in opportune positions on the two aromatic rings.
The general retrosynthetic scheme applied is reported in next Scheme 7.
The depicted synthetic approach is based on three principal reactions:
The main problem in the cyclization reaction is the low yields due to acid-catalyzed side reactions which lead complex mixture of byproducts [46][47][48]; this aspect is interesting from a kinetic point of view as recently reported [32].

4-and 5-substituted benzo[b]thiophenes as scaffold for benzothienopyridines
With these compounds in our hands we decided to use them as starting scaffolds for the synthesis of more complex heterocyclic structures. In particular, we focused on benzothienopyridines (Figure 2 a, b) with the aim to investigate new possible biological activities. In fact, it is well known that benzothienopyridines possess different pharmacological activities: their activity as antibacterial [50], antiallergic [51] and anxiolitic agents [52] were reported. Furthermore, pharmacological interest on benzothienopyridines is also due to their isosterism with indolopyridines [53,54]; recently also anti-cancer activity of such compounds has been reported [55].
Some of such reported compounds are currently in preclinical studies as inhibitors of BACE1 [62,64]: this fact confirms the great interest in the introduction of heteroatoms and heterocyclic fragments in the design of new structures.
To the obtained substituted benzothiophenes, the three steps synthetic scheme was applied and the results are reported in the schemes and tables (Tables 2-4).

Azido transfer reaction
Starting from the previous described 4-substituted-benzo[b] thiophenes, by using azidotransfer reaction, corresponding 2-azido heterocyclewere obtained in high yields due to the electron donating effect on the ring. As can be seen, the same groups in 5-position showed a smaller effect: chemical yields are lowered especially in the case of 2-azido compound with -OTBS (21) in 5 position, probably due to the less electronic enrichment on C-Li bond of thiophene ring.

Entry Azide
Yield Entry Azide Yield When we started from NHBoc-substituted benzothiophene the azido transfer reaction showed a drawback perhaps due to the presence of hydrogen on the aminic nitrogen: in fact, even by using excess of n-BuLi, azide yield on 4-NHBoc-benzothiophene (19) was very low (we hyphotized that NH could react during lithiation phase despite the steric hindrance of tert-butoxycarbonyl group).
However, the same reaction on 5-NHBoc furnish azide (22) in better chemical yields, probably due both to electronic effects than to an improved shielding effect of the NH.

Staudinger reaction
This reaction represents an interesting way to use a reactive function such as azido group in easy conditions by transforming in phosphorane, a very stable compound.
The best result in terms of yield has occurred in the case of iminophosphorane (23): Staudinger reaction starting from azide (17) with methyl(diphenyl)phosphine afforded the product (23) in 84% yield; in the case of the same reaction using triphenylphosphine, instead, there was a sharp drop in yield (24) [71,72].
The different reactivity of starting azide (17) is probably due both to electronic than to steric effects. The trend of reactivity is reversed starting from azide (20), which brings methoxyl group in 5-position, while the best result was observed for Staudinger reaction with triphenylphosphine (30). In the case of 4-OTBS derivatives the azide (18) showed less reactivity to Staudinger reaction, in particular with triphenylphosphine (iminophosphorane (26)), probably due to the steric hindrance either on the phosphorus than on the oxygen.
Unfortunately, iminophosphoranes resulting from 4-NHBoc derivatives were obtained only in traces, except for compound (33); no product was observed starting from 5-NHBoc-benzothiophenes (22) in the reaction with triphenylphosphine.
The trends observed can be explained on the basis of the mechanism of Staudinger reaction which initially involves the attack of the phosphorus on the azide to form a phosphazide intermediate as effectively reported in previous work [28]. If in the phosphazide the nucleophilicity of the nitrogen atom is not such as to favuor the intramolecular attack of the phosphorus, iminophosphorane formation may not occur.
In this view, the presence of electron releasing groups on the benzothiophene ring such as the methoxy and the OTBS favors this attack and consequently leads to higher yields in iminophosphorane; on the contrary, the presence of electron withdrawing groups, such as the Boc, disadvantage such attack. In fact, with the exception of iminophosphorane (33) obtained in 49% yield, in the other cases the   reaction did not take place (34) or led only to traces of the desired product (27,28).

Aza-Wittig/electrocyclization
The tandem aza Wittig/electrocyclization reaction represented the most important way to obtain pyridine and pyrimidine derivatives in good to excellent yields and in mild conditions. The reaction is well known in literature since 1970 and often used for the synthesis of different tricyclic compounds, such as benzothieno-, benzofuro-and indolopyridine.
The best result in terms of final yield (91%) is represented by benzothienopyridine (35), facile obtained in the cyclization of iminophosphorane (23). Also in this case, the effect of the presence of methoxy group on the benzothiophene ring was important and we a reverse reactivity of 5-substituted iminophospohoranes compared to the corresponding 4-derivatives can be observed.
On the other hand, the presence of a much more hindered group such as OTBS in 4-position results in a significantly lowering in product yield (36) starting from iminophosporane (25) and no cyclization product was observed starting from iminophosporane (26). Opposite effect is observed for 5-OTBS derivatives: in this case the desired product (39) was obtained in 13% yield starting from iminophosphorane (31) and in 58% yield starting from iminophosphorane (32). As regards NHBoc derivatives, electronic effects, which contrast/counteract not only to cyclization reaction but also to imine formation, play a crucial role; no cyclization products were observed in these cases.

Biological Essays
Beta secretase (BACE1) is an aspartyl protease that shows a crucial role in developing of Alzheimer Disease: in fact it seems plays a wrong cut of APP in neurons generating Ab-amiloyd peptide, which deposits out of neurons blocking the communication between this type of cells. The exact action 's mechanism of this enzyme is not yet well known but seems that its active site interacts with heterocyclic small molecules, such as some reported in previous  So we decided to test in vitro all the obtained benzothienopyridines for anti BACE 1 activity and some interesting value of IC 50 (inhibitor concentration needed to reduce enzyme activity by 50%) were obtained and reported in Table 5.
Compound 40 was obtained as byproduct in the Studinger reaction: even not belonging to the class of benzothienopyridines those smaller molecules were already tested on BACE1 to assess their activity. Compounds 42 and 43 were obtained after removal of TBS protecting group. Isomer 44 was obtained during the aza-Wittig electrocyclization step and isolated after purification. All compounds showed inhibitory activity values (IC50) in the range of micromolar scale. These promising results are comparable with the data of BACE1 known inhibitors [58][59][60][61]63] and actually are object of an Italian patent [74].

Conclusions
In this paper preliminary new heterocyclic compounds as inhibitors of BACE 1 are described. Among the reported compounds 35 and 42 showed higher inhibitory performance probably due to a better interaction of these molecules in the active site of the enzyme. The work is in progress to enlarge the library and to enrich the study of the interaction between inhibitors and catalytic site. Such research effort is oriented to obtained new and improved chemicals in Alzheimer's disease treatment. Obtained preliminary results encourage research advance and further developments.