Synthesis and Inhibitory Effect of Some Indole‐Pyrimidine Based Hybrid Heterocycles on α‐Glucosidase and α‐Amylase as Potential Hypoglycemic Agents

Abstract The Michael addition reaction of barbituric acid with chalcones incorporating the indole scaffold was achieved by using a highly efficient bimetallic Iron–palladium catalyst in the presence of acetylacetone (acac). This catalytic approach produced the desired products in a simple operation and low catalyst loading with acceptable yield of the new hybrids. All tested compounds were subjected for biological activity on α‐glucosidase and α‐amylase. The results revealed that all synthesized compounds exhibited very good activity against both enzymes when compared to positive control (acarbose). Moreover, compound 5o showed the best activity whereas its IC50 (μM) are 13.02+0.01 and 21.71+0.82 for α‐glucosidase and α‐amylase respectively. Both compounds 5o and 5l exhibited high similarity in binding mode and pose with amylase protein (4UAC). The obtained data may be used for developing potential hypoglycemic agents.


Introduction
Heterocyclic compounds are of immense chemical and biological significance. In particular, azaheterocycles (nitrogen containing heterocycles) such as pyrimidines and indoles are structural constituents of many natural as well as synthetic bioactive drug-like molecules. [1] Substituted azaheterocycles have been referred as "privileged structures" since they are capable of binding to many receptors with high affinity and hydrogen bonding capacity. Naturally occurring nitrogen-based heterocycles such as reserpine, vinca alkaloids, bisindoles, indoloquinolines, opioid analgesics, carbolines and cinchona alkaloids are established source of lead molecules for diverse therapeutic areas. [2] Among the nitrogen containing heterocycles, indole is the parent core in a large number of bioactive naturally occurring compounds. Indole and its derivatives have received significant attention due to their wide range of biological activities including antimicrobial, anticancer, anti-HIV antileishmanial and anti-inflammatory. [3] In recent past, several nitrogen containing novel chemical entities emerged as drug molecules, for example, Atevirdine (anti-HIV); Camptothecin (CPT) (inhibitors of topoisomerase I); [4] Cryptolepine (inhibitors topoisomerase II). [5] Synthetic analogues of Cryptolepine such as IQDMA and benzo-pyrido-indole derivatives exhibited potent anticancer activity via interaction of DNA [6] . We are engaged in a research program for drug development as anti-diabetes based on indole and pyrimdine scaffolds. [7] One example of our invention the use of indole scaffold in the treatment and prevention of diabetes has been described ( Figure 1). [7][8] Diabetes Mellitus (DM) is a growing global health concern. In 2017, diabetes affected an estimated 426 million adults people (20-79 years) world-wide; by 2045 this numbers are expected to overrun 629 million. [9] The release of free glucose from starch is mediated by two important enzymes: α-amylase and α-glucosidase. α-Amylase is a metalloenzyme that cleaves polysaccharide chains, semi-randomly creating shorter chains rapidly, whereas α-glucosidase breaks these shorter chains into free glucose. The inhibition of these two enzymes can delay digestion, and absorption of carbohydrates, and hence, impair ACN, or Toluene/THF mixture, the reaction did not occur. Other metal salt as Zn(OTf) 2 did not facilitate the reaction under the same conditions. Additionally, one attempt with FeCl 3 /PdCl 2 carried out in MeOH at 60°C, the reaction didnot occur at all.
Only, FeÀ Pd bimetallic system [11] in MeOH at 60°C provides the desired product in moderate yield (55 %) ( Table 1). The molecular structures of target compounds 5a were determined by analysis of its spectroscopic data including 1 H-, 13 C-NMR, Fourier-transform infra-red (FT-IR) spectroscopy and X-ray crystal analysis.
To investigate the generality of this method, the reaction of barbituric acid and different enones was examined under the optimized reaction conditions (10 mol% of FeCl 3 , 10 mol% of PdCl 2 and 15 mol% Acac, 1.0 equiv. chalcone and 1.1 equiv. barbituric acid in CH 3 OH at at 60°C. All of the results are summarized in Table 2.

X-Ray Crystallography
The structure of 5g was further confirmed by X-Ray structural study. The asymmetric unit contains one independent molecule that is shown in Figure 2. It was found to crystallize in Monoclinic Cc space group. The crystallographic data and refinement information are summarized in Table 3 and bond lengths are in normal ranges as shown in Table 4. The crystal structure reveals that the title compound is found in three planes, the angles between indole ring plane (C1À C8/N1) and fluorophenyl ring (C12À C17) and pyrimidine moiety (C20À C21À N2À C22À N3À C23) are 22.41°and 41.07°, respectively. The angle between fluorophenyl ring and pyrimidine ring is 57.88°. The crystal structure is stabilized by many non-classical hydrogen bonds along the b axis direction Figure 3, Table 5.

Biological Activity
The present study seeks an alternative drug among series of synthesized compounds that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by using virtual and in vitro assays. The data reported in Table 6 showed that the most active compounds, both on α-glucosidase and on α-amylase, are compounds 5o, 5k, and 5l. All other compounds were found to have only good to moderate activity ranging from 28.05 + 0.41 to 77.05 + 0.04 μM in the case of α-glucosidase, but in the range of 53.10 + 0.10 to 96.42 + 0.22 μM in the case of α-amylase. Structure activity relationship indicates the importance of the naphthyl moiety in 5o, of the p-CF 3 Ph propanone substituted indole in 5k, and of a thiophene ring in 5l. The most active compound is 5o, which showed an IC 50 = 13.02 + 0.01 μM and 21.71 + 0.82 μM, for α-glucosidase, and α -amylase respectively.

Docking Studies
The compound 5 o was selected for docking study with (4UAC) because of its strongest inhibitory activity among these derivatives. The X-ray crystal structure of (4UAC) was obtained from protein data bank (PDB ID: 4UAC). [12] Protein-ligand docking was operated by (OpenEye Scientific Software, Santa Fe, NM 87508). [13] The binding site of the protein was prepared by employing FRED RECEPTOR 2.2.5 (OpenEye Scientific Software, Santa Fe, NM 87508).
In the figure 4, we can find that compound 5o formed hydrogen bonds to ASN 191 AA through the oxygen of carbonyl linked to indole moiety. Moreover, this compound formed another HB with GLN 110 AA through the carbonyl of barbiturate ring. These two interactions are similar to acrabose standard with receptor in its cocrystalized from. [12] Compound 5o exhibited high similarity to the potent derivative (compound 5l) in the specific receptor, figure 5.

Experimental Section
General Procedure for the Synthesis of Chalcones 3a-q The chalcones were prepared followed by reported procedure. [14] (

Docking Studies
A virtual library of designed compounds was energy minimized using the MMFF94 force field, which was followed by the generation of multi-conformers using the Omega application. The entire energy-minimized library was docked with the prepared catalytic domain of (PDB code: 4UAC) [12] using the FRED application in OpenEye software [13b] to generate a physical property (ΔG) reflecting the predicted energy profile of the ligand-receptor complex. The Vida application can be employed as a visualization tool to show the potential binding interactions of the ligands with the receptor of interest.

Conclusion
The present study mainly focuses on the synthesis of novel indole-pyrimidine based chemical entities for the improved anti-diabetic activity. The new series of indole-pyrimidine based compounds obtained via bimetallic catalytic system which has a dramatic effect in promoting the Michael addition reaction. The synthesized compounds screened against wide range of α-glucosidase inhibition and α -amylase assay inhibitory activity. Docking study describes that both barbiturate and acyl indole parts participate in HB while the aryl linker occupy the receptor through lipophilic-lipophilic interactions.