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Insulin-regulated aminopeptidase (IRAP)
Insulin-regulated aminopeptidase (IRAP) is a type II transmembrane protein belonging to the family of zinc-dependent membrane aminopeptidases. This enzyme was originally cloned and characterized in glucose transporter type 4 (GLUT4) vesicles of muscle and fat cells. GLUT4 is a protein which regulates glucose homeostasis. At elevated levels of insulin, IRAP accompanies GLUT4 to the plasma membrane to normalize the level of glucose. IRAP was found have three domains: a large extracellular catalytic domain, a single transmembrane region and an intracellular domain.
Ang IV binding site was originally referred to as the angiotensin IV (AT4) receptor, but in 2001 AT4 receptors in bovine adrenal membranes were purified, sequenced and determined to be IRAP.32 Likewise, the endogeneous peptide LVV-hemorphin 7 (LVV-H7) has also been described as a ligand to IRAP. These two neuropeptides are said to be competitive inhibitors of IRAP, where they bind at the catalytic site of the enzyme thereby inhibiting its activity.32-33 The mechanism through which IRAP promotes memory is not clear, though one theory states that the binding of these ligands increases the half-life of IRAP substrates such as, vasopressin, oxytocin and somatostatin which are known to play a major role in enhancing learning and memory.
Peptide inhibitors
The discovery of IRAP initiated studies wherein, Ang IV was modified through processes such as truncations of peptide bonds, macrocylization and introduction of conformational strains at different amino residues. One of these peptidomimetics IRAP inhibitors (HA-08), synthesized via introduction of constrained macrocyles, exhibited potency 20 times that of Ang IV (Figure 1). The disadvantage of these inhibitors is that they are susceptible to degradation and may not cross the blood brain barrier.
Non-peptide inhibitors
The limited use of peptidic inhibitors has led to other methods being used to generate IRAP inhibitors, one being structure-based design and receptor based virtual screening.36 Structure- based design and receptor based virtual screening is a method of designing a drug based on information about the target (protein) compound with help of a computer.37 Targets are first identified and their 3D structures determined by X-ray crystallography (XRC) or nuclear magnetic resonance (NMR).37-38 In cases where targets are not known, prediction through homology modeling can be used to generate one.39 Virtual screening of large library compounds against the indentified target to obtain hit compounds follows. The hit compounds are thereafter purchased or synthesized, tested in bioassays and further optimized to come up with powerful inhibitors.36-38 Albiston et al., used this approach to identify and design benzopyran-based IRAP inhibitors. A total of 1.5million commercially available compounds were screen against a homology model of IRAP. One of the inhibitors (Figure 2) obtained after optimization of the hit compound, demonstrated memory enhancing effect in rats.
Microwave-assisted synthesis
In this project a microwave equipment was used to perform the synthesis. Microwave-assisted synthesis has transformed synthetic chemistry since its implementation in the mid 80s.
Compared to conventional heating such as oil bath, this technique offers the following advantages: increased rate of reaction because higher temperatures can be used, cleaner reactions and higher yield due to less side reactions and reduced consumption of energy since microwaves heat up the sample and not the reaction vessels.44 Microwaves lie between 0.01 m to 1 m in the electromagnetic spectrum (EMS), corresponding to a frequency of 30 GHz to 0.3 GHz. The reaction heating is nevertheless done at 2.45 GHz.43-44 Substances with dipole moment, polar or ionic solvents are suitable for use in the microwave, because when irradiated they can generate heat through mechanisms like conduction and dipolar polarization.45 Microwave synthesis apparatus can have single- or multi-mode cavity. A single mode oven accommodates one vessel at a time, while multiple modes cavities can run many samples at a go.44 Reaction vessels are always transparent to microwave and are made of materials such as borosilicate glass or teflon.43.Some recent advances in microwave–assisted synthesis include enhanced microwave synthesis46 and microwave heated flow synthesis.
N-methylatation of isatins
The methylations of the isatins follow the SN2 reaction mechanism. SN2 is a reaction involving a nucleophile and an electrophilic substrate. A nucleophile is a negatively charged ion or a neutral molecule with unshared electrons, which seeks the positive center in a substrate in a reaction. A substrate contains electrophilic centers with low electron density and a leaving group (LG). LG is a substituent that leaves as a stable molecule or ion during a nucleophilic attack. Halogens in alkyl halides are good LG because they can leave as weak bases and stable ions. Factors that favor SN2 reactions are unhindered substrate, polar aprotic solvents and strong nucleophiles. Substrates with bulky substituents will hinder the reaction, while polar protic solvents can protonate the nucleophile thereby decreasing its reactivity. The rate of reaction is increased by concentration of both the nucleophile and the substrate.
Reaction mechanism for the N-Methylation of isatin
The reaction starts with the deprotonation of the halogenated isatin (2) by the base Cs2CO3, generating the isatin anion (2-). This anion then acts as a nucleophile, attacking the electrophilic carbon of iodomethane and at the same time iodine leaves and compound 3 is formed (Scheme 1).
Method and Results
The synthetic route for the alkylations and the results are displayed in table 1. Compounds 3a, 3b and 3c were obtained in good yields and used in the next step without further purification. Compound 3c was impure and had to be purified. The purification was challenging because a solvent system suitable for the separation was not obtained. Hence, only a portion of it was isolated after purification leading to a lower yield.
Reaction mechanism
The reaction mechanism for synthesizing the halogenated derivates (6a, b, d) and other analogues of 1 (6e-f) which are synthesized in this project, proceeded through the following steps (Scheme 3): An acid catalyzes the reaction by protonating one of the carbonyl oxygen of 4, making it more electrophilic. This process facilitates the neucleophilic attack by 5, followed by a decarboxylation to form the intermediate (8). Another protonation ensues, this time on 3. This makes it possible for 8, which is equipped with a neucleophilic primary amine to attack the most electrophilic carbon in 3 and water is eliminated. An imine is subsequently formed by the reaction of the primary amine in 8 and the ketone in 3. The imine undergoes intramolecular cyclization to give 6.
Optimization results and discussion for the synthesis of 6e
Table 3 gives the results of the optimization procedures attempted. From the output, it is clear that a method that could give a pure product in good yield was not obtained in this study. An attempt was made to isolate and purify the traces of product seen in method 2 and 3, via flash column chromatography. The process turned out to be challenging, because the number of spots on the TLC plate, together with their position to each other (Rf range less than 0.2-0.3), made it difficult to come up with a suitable solvent system to use as an eluent. To succeed with this isolation, repeated purifications needed to be done and this would result in poor yields. Due this reasoning, methods 2 and 3 were not further purified and were hence abandoned.
It was also not possible to establish an optimum temperature or time for the all the reactions carried out in the microwave, because increasing reaction time (from 10 min to 40min) or temperature (from 120oC to150oC) did not result in complete consumption of the limiting NHOOONOOBr++MW,SolventtempCatalyst6ONNHNOBrRisatin (3c, 1 equiv)isatoic anhydride (4, 1,2 equiv)amine(5, 1.2equiv)RNH2Scheme 4: Synthetic route and equiv amounts used for synthesis of 6e-f reactant (3c). Its peak was still visible in the LC-MS report and this was going to impact on the yield had the methods been used.
Since the microwave reactions were unsuccessful, conventional reflux heating were tried (method 7 and 8). The results were not any better. The reactions were not clean, had multiple by-products and were not going to be easy to handle during the workup and purification steps.
Important information gained from the methods in table 6 was the presence of 8b (intermediate) at the end of most of reactions. The peak of this intermediate was more pronounced compared to other peaks in several LC-MS reports. This gave the view that not much reaction was taking place after the formation of this intermediate. Thus, a decision was made to carry out the reaction in two steps. The first step involved synthesizing 8b, then using it in the next step to form 6e.
Table of contents :
1. Introduction
1.1. Cognitive disorder
1.2. Alzheimer’s disease
1.2.1. Etiology and pathogenesis of Alzheimer’s disease
1.3. Current available drugs for treating Cognitive disorder
1.3.1. Cholinergic system
1.3.1.1. Cholinesterase inhibitors
1.3.1.2. Ach receptor modulators
1.3.2. Glutamatergic system
1.3.2.1. NMDA antagonist
1.4. Background to the present study
1.4.1. Angiotensin (IV)
1.4.2. Insulin–regulated aminopeptidase (IRAP)
1.4.3. Peptide inhibitors
1.4.4. Non-peptide inhibitors
1.4.5. HTS
2. Aim of the present study
2.1. Structural modifications
3. Microwave-assisted synthesis
4. N-methylation of isatins
4.1. SN2 reaction
4.2. Reaction mechanism for N-methylation of isatins
4.3. Method and Results
5. Synthesis of the halogenated analogues
5.1. Imine formation
5.2. Reaction mechanism
5.3. Method
5.4 Results and discussion
6. Other modifications
6.1. Method development
6.2. Optimization results and discussion for the synthesis of 6e
6.3. Optimization results and discussion for the synthesis of 8b
6.4. Results for the synthesis of 8b-d using the method developed
6.5. Optimization results and discussion of the synthesis of 6
6.6. Results for the synthesis of 6e using the method developed
6.7. Results for the synthesis of 6e-f using the method developed
7. Biological evaluation
7.1. Initial SAR study results
7.2. Biological assays results
8. Conclusion and outlook
9. Acknowledgements
10. Experimental data
11. References
