Blonanserin N-Oxide Lowers Glucose Levels in Animal Models

The N-oxide of anti-psychotic drug Blonanserin (BLNO) was synthesized and tested as a novel molecule to understand its ability in reducing blood glucose levels. Administration of BLNO at 1 mg/kg (mpk, mg of drug per kg of rat body weight) to diabetic Wistar rats (induced by treatment of 40 mg/kg streptozotocin, STZ) showed reduced glucose levels at 100 mg/dL in a 28 day study, compared to the control group (with no drug added only STZ induced) which reached hyperglycemic levels of 500-600 mg/dL. At 5 mpk of BLNO, glucose levels were controlled in a 18 day study and no toxic or adverse indications were observed at the higher 5X dose. Oral glucose tolerance test on Wistar rats confirmed the potential of BLNO to reduce glucose levels at 5 mpk dose, comparable to that of marketed drugs metformin HCl (MHCL) and sitagliptin phosphate monohydrate (SPMH) at 100 mpk dose in 90-120 min after drug administration. A novel molecule Blonanserin-N-oxide is identified as a potential anti-diabetic drug lead for controlling blood glucose levels.

Keywords: Diabetes; Glucose; Blonanserin; N-Oxide; GPCR; Antipsychotic

Type-2 Diabetes mellitus (DM) is a metabolic disorder caused by insulin resistance (dysfunction of pancreatic beta cells) and obesity. It is now recognized as a major health problem worldwide and affects adults of working age in developing countries. WHO estimates on the global prevalence of diabetes are expected to increase from 171 million in 2000 to 366 million in 2030. As a matter of fact 21.7% of these (~80 million) will be from India [1]. A first-line treatment for diabetes is lifestyle changes such as low calorie diet, weight control, and regular physical activity. If blood sugar (glucose) levels still remain high, then the patient is administered anti-diabetic drugs (for Type-2 diabetes) or insulin (Type-1 diabetes mellitus). A major challenge in T2DM therapy is how to make the pancreas more insulin sensitive. The other goal is to increase endogenous insulin levels in T2DM patients. One way to address the problem is to target pancreatic β-cells for increasing endogenous insulin levels via small molecule drugs (bigunides, sulfonylureas, meglitinides, thiazolidinediones, alpha-glucosidase inhibitors, dipeptidyl-peptidase IV inhibitors, incretin-based therapies, etc.) [2-4]. Metformin (bigunide), is the classic example of a T2DM drug which is an insulin sensitizer. However metformin requires circulating insulin levels for drug action but does not activate insulin secretion. The sulfonylureas, or ATP channel blockers, gliclazide, glimepiride, glipizide, tolbutamide, etc. promote the release of insulin from the β-cells by blocking the ATP-sensitive K+-channels, thereby resulting in depolarization with Ca+2-influx which activates insulin secretion. However, a serious side-effect of the sulfonyl urea drugs is hypoglycaemia, which happens due to excess insulin release. This means that the patient has to have additional sugar or carbohydrate to normalize insulin levels. The dipeptidyl peptidase IV inhibitors (also known as incretin enhancers or DPP-IV inhibitors, [5] are the latest generation T2DM drug molecules, e.g. sitagliptin, linagliptin, saxagliptin, and vildagliptin [6]. The glucagon like peptide (GLP-1 and 2) are natural incretin hormones which are released from the gut in response to food intake, and they are collectively responsible for glucose-dependent insulin release [7]. The physiological activity of the incretin hormones is limited by DPP-IV peptidase enzyme which cleaves the terminal end of active GLP to inactive peptide. This is reversed by the gliptin drugs, which are tight binding inhibitors of the DPP-IV enzyme. An advantage of the gliptins, compared to say the glitazones, is that since GLP levels are modulated by food intake, insulin release occurs post prandial and hence hypoglycemic condition is avoided. However, complete studies on the potential side effects of incretin-based gliptins are still ongoing [8].

There is increasing evidence in the past decade that patients taking anti-psychotic drugs for schizophrenia run an increased risk of diabetes mellitus [9]. That anti-psychotic drugs cause weight gain, and together with reduced physical activity in such patients, are additional factors for the high incidences of T2DM in psychotic patients [10,11]. A recent study showed that phosphorylation of M3-muscarinic receptor plays an important mechanistic role in facilitating insulin release from pancreatic islets by coupling of the receptor to protein kinase D1 [12]. These muscarinic acetylcholine receptors are a subfamily of GPCRs recognizing the neurotransmitter acetylcholine and signaling through G proteins of the Gq/11 class. These receptors are also targets for the treatment of diabetes and have been implicated in the treatment of cognitive disorders such as Alzheimer’s disease and schizophrenia.

The N-oxide of anti-psychotic drug clozapine (CLNO, clozapine N-oxide) is a natural metabolite and it was studied in transgenic mice for the activation of β-cell signaling pathway in vivo [13]. When transgenic (Tg) mice were injected with glucose alone, the blood glucose and insulin levels remained similar to the wild type set. However, co-injection of glucose (3 g/kg, i.p.i.p.) resulted in an immediate increase in insulin release (2 min) and subsequent time points (up to 15 min), at levels almost 12 times higher than the first 10 min, compared to the control group. There was a two-fold increase in islet density and 30% increase in β-cell mass size after a 2 weeks treatment [14]. Thus the pharmacological effects of CLNO are fundamentally different compared to previous drug classes, in that it not only activates insulin secretion instantaneously but also increases the size and mass of the pancreatic islet β-cells [15]. In the same class of anti-psychotic drugs is blonanserin (BLN), an agent with dopamine D2 and serotonin 5-HT2A receptor antagonist properties, [15] similar to SNRI drug clozapine (CLN) (serotonin norepinephrine reuptake inhibitors); both drugs are used in the treatment of schizophrenia and central nervous system (CNS) disorders [16]. Based on the structural similarity of these two molecules (Figure 1), we undertook the synthesis of the natural metobolite BLNO and to screen its anti-diabetic activity.

Blonanserin was purchased from Mesochem Technology Co. Ltd., China, and used without further purification. Clozapine was purchased from Shanghai Xunxin Chemical Co Ltd, China. All other chemicals and reagents were purchased from domestic suppliers and are of analytical grade purity. Metformin hydrochloride (MHCL) and Sitagliptin phosphate monohydrate (SPMH) were purchased from drugs suppliers. Streptozotocin and solvents and reagents were purchased from Sigma-Aldrich and Merck (Hyderabad, India).

Blonanserin (200 mg, 0.54 mmol) was dissolved in 15 mL of methanol and stirred at room temperature until it dissolved completely. To this reaction mixture 1.44 mL of 30% H₂O₂ was added and refluxed at 70 ^°C for 2 h. After completion of the reaction (by TLC), methanol was removed by rotary evaporator resulting in a viscous liquid. The reaction mixture was diluted with water and extracted with CHCl₃. Drying of the organic layer with Na₂SO₄ evaporation of volatiles and drying on a vacuum pump for 2 h afforded a white solid which was purified by recrystallization from n-pentane. Yield 150 mg (70%); m.p. 153-155 ^°C (decomposition) [17]. 30 mg of this BLNO was dissolved in 5 mL DMSO: H₂O (1:1 v/v) and left for slow evaporation at room temperature. Good quality single crystals were harvested after about one week for single crystal X-ray diffraction analysis.

¹H NMR (CDCl₃, 400 MHz): 7.22 (t, J 12 Hz, 2 H), 7.10 (t, 12 Hz, 2 H), 6.37 (s, 1 H), 4.13 (d, 11 Hz, 2 H), 3.82 (t, 8 Hz, 2 H), 3.50-3.35 (m, 2 H), 3.35-3.20 (m, 2 H), 2.90 (d, 4 Hz, 2 H), 2.60 (t, 8 Hz, 2 H), 1.80 (m, 3 H), 1.50-1.36 (m, 10 H).

IR (KBr, cm^-1): 3412(br.), 2928, 2853, 1588, 1542, 1502, 1458, 1413, 1222, 1155, 1093
HRMS: m/z 384 [M+1] peak

X-ray reflections were collected on Oxford CCD X-ray diffractometer (Yarnton, Oxford, UK) equipped with Mo-Kα radiation (λ = 0.71073 Å) and Cu-Kα X-radiation (λ = 1.5406 Å) source. Data reduction was performed using CrysAlisPro 171.33.55 software. Crystal structures were solved and refined using Olex 2-1.0 with anisotropic displacement parameters for non-H atoms. Hydrogen atoms were experimentally located through the Fourier difference electron density maps in all crystal structures. All O–H and C–H atoms were geometrically fixed using HFIX command in SHELX-TL program of Bruker-AXS. X-Seed was used to prepare packing diagrams.

Empirical formula: C₂₃H₃₄FN₃O₃, Formula weight: 473.5, Crystal system: Orthorhombic, Space group: Pbca, T = 298 K, a = 8.8000(7); b=13.9995(15); c=36.033(3) Å, α = β = γ = 90^°, Z = 8, Z^'= 1, V = 4439.1(7) ų, R-factor = 0.0734 [18].

Clozapine (200 mg, 0.61 mmol) was dissolved in 4 mL of dichloromethane and kept at 0 ^°C for 5 min. To the cooled solution, m-CPBA (180 mg, 1.15 mmol) as a solution in CH₂Cl₂ was added drop wise at 0 ^°C. After 1 h, TLC indicated that the reaction was complete. NaHCO₃ solution was added and stirred for 10 min. The reaction mixture was extracted with CHCl₃ and the organic layer was dried with Na₂SO₄. Evaporation of the solvent afforded a light yellow solid which under vacuum for 2 h. Purification by recrystallizaton from n-hexane afforded CLNO. Yield: 160 mg (80%), m.p. 190-200 ^°C (dec.). Clozapine N-oxide was also oxidized using 30% H2O2 also in reasonable yields.

¹H NMR (CDCl₃, 400 MHz): 7.25 (dd, 8, 2 Hz, 1 H), 7.15 (dd, 8, 2 Hz, 1 H), 6.95 (m, 2 H), 6.80 (d, 6 Hz, 2 H), 6.70 (d, 6 Hz, 1 H), 5.20-5.10 (m, 1H), 4.00-3.80 (m, 2 H), 3.50-3.30 (m, 2 H), 1.20 (s, 3 H).