Targeting PPAR γ Receptor Using New Phosphazene Derivative Containing Thiazolidinedione: Design, Synthesis, and Glucose Uptake

The peroxisome proliferator activator receptor-γ (PPAR-γ) remained the most effective target for management of diabetes mellitus. The present work endeavors rational designing new PPAR-γ agonist bearing cyclotriphosphazene and thiazolidine-2,4-dione scaffolds. Thiazolidinedione (TZD) derivatives are the novel class of oral antidiabetic drugs which are selective agonist for the nuclear PPARγ that enhances the transcription of several insulin responsive genes but TZDs are known to cause weight gain, hepatotoxicity and fluid retention. So, cyclotriphosphazene containing thiazolidine-2,4-dione was de-signed, synthesized as PPARγ agonist. The in-vitro antidiabetic activity showed that compound 8 has similar activity and exhibited higher glucose uptake in comparison to pioglitazone as reference drugs. This research opened new avenues for smart designing of molecules with high efficiency towards the management of hyperglycemia.

is one of the major health problems in the world today. The incidence of the disease currently is estimated to reach 300 million by the year 2025. Most cases will be of Type 2 diabetes mellitus, which strongly linked with a sedentary life style and obesity. Recently, chemistry of 2,4-thiazolidinediones (TZDs) has attracted attention as they have been found to exhibit several biological activities, such as ntihyperglycemic, anti-inflammatory, antimalarial, antioxidant, antitumor, cytotoxic, antimicrobial, and antiproliferative. Thiazolidinediones (TZDs), which are known to sensitize tissues to insulin, have been developed and clinically used as antidiabetic agents. They have been shown to reduce plasma glucose, lipid, and insulin levels, and used for the treatment of type 2 diabetes [2].
As agonists of nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), thiazolidinediones (TZD) reduce insulin resistance in the liver and peripheral tissues; increase the intake of insulin-dependent glucose and decrease withdrawal of glucose from the liver [3]. Many drugs have been approved from this class for the treatment of diabetes like Rosiglitazone, Pioglitazone, Ciglitazone and many more. Though the marketed drugs show additive effect with other antihyperglycemic agents, they are also prone to show toxicity.
Phosphazenes are class compounds with interesting properties. They showed a number of characteristics such as biomedical properties and applications due to their strong antitumor activity [5]. Their antimicrobial and biological activities on bacterial and yeast cells have been studied [6]. A variety of substitution reactions of the reactive P-Cl bonds provide a wide range of cyclophosphazene derivatives, which have diverse applications [7]. These derivatives are usually synthesized by nucleophilic substitution reactions using alcohol, phenol, amines, Grignard reagents and thiols [8]. Their physical and chemical properties can be tailored by appropriate substituents on the phosphorus atoms.
Hence, there is a need for the development of newer and safer drugs from this class. There is still an urgent need for novel anti-diabetic agents that should have a similar degree of efficacy with a potential to reduce long-term complications. In our efforts to develop the biological profile of these analogues, we have reported an efficient synthesis and screening of new thiazolidinedione derivative as potential antidiabetic drugs. Inspired by the diverse biological properties of thiazolidinedione moiety and cyclotriphosphazene, in the present study, an attempt has been made to synthesize title compound by employing hybridization approach with the hope that the resulting new molecules will have anti-diabetic activity. The structures of the synthesized compounds were assigned based on elemental analysis, IR, 1 H, 13 C and 31 P NMR spectroscopy. The compound was also screened for their in-vitro antidiabetic activity.

Chemistry
All reagents and chemicals were purchased from Sigma-Aldrich and used with-Open Journal of Medicinal Chemistry out further purification. Thin-layer chromatography (TLC) was performed on silica gel glass plates (Silica gel, 60 F 254 , Fluka) and the spots were visualized under a UV lamp. Column chromatography was performed on a Kieselgel S (silica gel S, 0.063 -0.1 mm). The melting points were recorded on a Gallenkamp apparatus and were uncorrected. Infrared spectra were measured using KBr pellets on a Thermo Nicolet model 470 FT-IR spectrophotometer. 1 H-NMR spectra were recorded on 400 MHz Varian instruments using DMSO-d 6   tonitrile, kept at 0˚C, K 2 CO 3 (5 mmol) was added. The reaction was stirred on the ice-bath for 5 minutes then the vessel was closed immediately and was subjected to microwave irradiation at 140˚C for about 30 min. After cooling to r.t, 20 mL of ethyl acetate was added and solution was neutralized with drop wise addition of ~6 M HCl (pH = 7 -7.5), washed with a saturated NaHCO 3 and NaCl solution (1:3, 1 × 50 mL). Aqueous layer was rewashed with ethyl acetate (2 × 25 mL) and the combined organic layer was dried. The product was purified by silica gel column chromatography with hexanes/dichloromethane/ethylacetate as an eluent to give 5. This compound was white powder, yield 89%; mp: 224˚C;

Synthesis of Phosphazene-Thiazolidinedione (8)
A mixture of N-methylthiourea 6 (3.0 mmol, 0.27 g), chloroacetic acid 7 (3.6 mmol, 0.2 ml) and aldehyde 5 (3.0 mmol, 0.74 g) was heated under microwave irradiation at 90˚C -110˚C for 10 -20 min. After cooling to room temperature, the reaction mixture was extracted with CH 2 Cl 2 . The organic layer washed with aquous NaHCO 3 , water and dried over anhydrous Na 2 SO 4 . The solvent was removed under vacuum and the residue was recrystallized from EtOH/water to give 7 as pale yellow powder; yield 90%; mp 213˚C; IR (KBr, cm  was 216 p mol/L. The average intra and inter assay coefficients of variation were 3.37% and 2.29%, respectively. The levels of insulin were expressed as p mol/L.

Statistical Analysis
Experimental results were expressed as mean ± SEM and statistically assessed by SPSS-20. The difference between test animals and control was evaluated using the Student t-test.

Chemistry
The synthesis to produce the dioxybiphenyl derivative 3 via the reaction depicted in Figure 1. Starting from hexachlorocyclotriphosphazene 1, two equivalents of bip-henyl-2,2'-diol 2 were added to four equivalents of potassium carbonate. In this case, only two equivalents of the reagent are needed as one bip-henyl-2,2'-diol is capable of bonding to one phosphorus atom through the two deprotonated oxygen molecules. Two phosphorus atoms are then substituted with this bulky group. This reaction was performed in acetone as the reaction has been proven Open Journal of Medicinal Chemistry to occur faster in acetone than in THF [9]. It was also not necessary to reflux the first step in this reaction as this bifunctional nucleophile promotes the replacement of the chlorine atoms. In fact, the trimer and biphenol were added to acetone that was cooled in an ice bath. This is presumably done to prevent the reaction from taking place too fast and forming trimer that is triply substituted with bip-henyl-2,2'-diol [9]. When the first nucleophilic oxygen reacts with the phosphorus atom, it activates the phosphorus atom for further substitution (geminal substitution) [10]. The neighbouring oxygen is the next-closest nucleophile and substitution of the second chlorine atom occurs. Crosslinking of trimer molecules does not occur because the closest reactive site is on the same molecule [9]. The reaction mixture was stirred at room temperature for 24 hours under an atmosphere of dry nitrogen because bip-henyl-2,2'-diol is air sensitive. The solvent was then removed under vacuum and the product extracted with DCM. More than one product can possibly form during the reaction, but the main product is isolated via extraction and recrystallisation as the solubility of the doubly and triply substituted derivatives differs in different solvents. The reaction depicted in Figure 2 was carried out by microwave in which 2 equiv. of 4-hydroxybenzaldehyde reacted with 3 in the presence of K 2 CO 3 in acetonitrile for 30 min at 140˚C gave 2,2-bis(4-formylphenoxy)-4,4,6,6-bis [spiro(2',2''dioxy-1',1''-biphenylyl)]cyclotriphosphazene 5. It is necessary to increase temperature in this step as the groups on the cyclotriphosphazene ring are quite bulky, causing steric hindrance which would mean that the reaction will need more energy to take place. It was not necessary to do this reaction under inert conditions. The work-up for the second step is similar to the first step. The solvent was removed under vacuum and the product extracted with dichloromethane (DCM). The DCM was then removed in vacuo.
The new phosphazene derivative containing a thiazolidine-2,4-dione 8 was synthesized from the reaction of 5 with N-methyl thiourea and monochloroacetic acid under microwave-irradiation for 15 min succeeded to afford the desired product 8 in 90% yield. Compound 8 was characterized by elemental analysis, FT-IR, 1 H, 13 C, 31 P NMR techniques (Figure 3).
The structure of the obtained compound was established based on their elemental analysis together with their compatible spectra data. The IR spectra of compound showed characteristic absorption bands at 3423 cm −1 and 1688 cm −1 corresponding to the -NH and C=O groups in the obtained structure. Another band at 1503 cm −1 attributed to C=N group. In addition, the 1 H-NMR spectrum Open Journal of Medicinal Chemistry   Figure 4). The 31 P NMR spectrum of 8 two signals were observed as one doublet and one triplet, which indicates that the two phosphorus atoms attached to the dioxybiphenyl ring are not magnetically equal. This non-equivalence of the two phosphorus atoms maybe due to the difference in the angle of twist of the two phenyl groups of the biphenyl moieties and their twistss in a different direction. The reason for this reversal twist/distortion could be due to the advantageous thermodynamically stable seven-membered dioxybiphenyl ring conformation by imparting reduced 6,6' hydrogen-hydrogen contacts without broadening the O-P-O angle.

Anti-Diabetic Activity
Peroxisome proliferator-activated receptors (PPARs) are known transcription factors that directly control the expression of genes involved in lipid and glucose metabolism [11]. The mechanism of PPARs has been described [12]. Among the Open Journal of Medicinal Chemistry three isotypes of PPARs (PPARα, PPARβ and PPARγ), PPARγ is the most studied for drug discovery. PPARγ was not only identified as a key regulator of adipogenesis, but it also plays an important role in type 2 diabetes, cellular differentiation, insulin sensitization, atherosclerosis and cancer [13]. A class of highaffinity PPARγ synthetic ligands includes the anti-diabetic thiazolidinedione (TZD) drugs, such as troglitazone, rosiglitazone, pioglitazone and ciglitazone [14]. Rosiglitazone and pioglitazone are currently marketed PPARγ activators used for the treatment of type 2 diabetes to reduce hyperglycemia by promoting insulin action without additional insulin secretion [15]. TZD-type improves insulin resistance with side effects like weight gain, fluid retention and edema. In the present study, a novel and effective thiazolidinedione-2,4-dione derivative have been synthesized as potential antidiabetic drugs that may bind and activate PPARγ and enhances insulin sensitivity. An in-vitro study showed that the new thiazolidinedione-2,4-dione derivative provides a new insight concerning their effect on PPARγ.

Effect of Compound 8 on Insulin Secretion from βTC6 Cell Line
Secretion of insulin by βTC6 cells was measured using the high range insulin Sandwich ELISA kit. Figure 5(a) shows the effect of pioglitazone on insulin secretion in the presence and absence of 2.88 mM glucose. As can be seen from Figure 5

Glucose Uptake Assay
The results of the in vitro glucose uptake study indicate that the compound 8 was found to exhibit remarkable potential to flush glucose into the mentioned cells as compared to standard reference Pioglitazone. Compound 8 happens to be a potent compound by enhancing the glucose uptake significantly (P < 0.05) ( Figure 6).