Novel Liver-targeted conjugates of Glycogen Phosphorylase Inhibitor PSN-357 for the Treatment of Diabetes: Design, Synthesis, Pharmacokinetic and Pharmacological Evaluations

PSN-357, an effective glycogen phosphorylase (GP) inhibitor for the treatment for type 2 diabetics, is hampered in its clinical use by the poor selectivity between the GP isoforms in liver and in skeletal muscle. In this study, by the introduction of cholic acid, 9 novel potent and liver-targeted conjugates of PSN-357 were obtained. Among these conjugates, conjugate 6 exhibited slight GP inhibitory activity (IC50 = 31.17 μM), good cellular efficacy (IC50 = 13.39 μM) and suitable stability under various conditions. The distribution and pharmacokinetic studies revealed that conjugate 6 could redistribute from plasma to liver resulting in a considerable higher exposure of PSN-357 metabolizing from 6 in liver (AUCliver/AUCplasma ratio was 18.74) vs that of PSN-357 (AUCliver/AUCplasma ratio was 10.06). In the in vivo animal study of hypoglycemia under the same dose of 50 mg/kg, conjugate 6 exhibited a small but significant hypoglycemic effects in longer-acting manners, that the hypoglycemic effects of 6 is somewhat weaker than PSN-357 from administration up to 6 h, and then became higher than PSN-357 for the rest time of the test. Those results indicate that the liver-targeted glycogen phosphorylase inhibitor may hold utility in the treatment of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) continue to expand at epidemic rates and new medicines targeting novel mechanisms are urgently needed. Glycogen phosphorylase (GP) is the key enzyme that catalyzes glycogenolysis, leading to the release of glucose from glycogen 1 . Three isoforms of GP have been identified that located within different metabolically active tissues for different physiological functions 2 . The muscle isoform provides energy for muscle contraction, the brain isoform provides an emergency supply of glucose during periods of anoxia or severe hypoglycemia, and the liver isoform regulates glucose release from hepatic glycogen stores 3 . In addition, it has been demonstrated that the inhibition of GP is involved in the promoting of glycogen synthesis in liver. Thus, GP, especially, the isoform in liver has received great recent interest as potential target for T2DM 4 . Although liver GP inhibition is regarded as an excellent therapeutic target for the treatment of diabetes, one very important factor relating to the relevance and importance of isoform specificity with this new therapeutic remains to be proven. As previously stated, brain, liver and skeletal muscle isoforms demonstrate 80% homology in their structures 5 , thus finding 100% specific inhibitors of the liver isoforms has proved difficult. Therefore, drug development programmes must consider the potential side effects of such compounds in relevant models. For example, inhibition of skeletal muscle GP when the liver isoform is the primary target could have devastating effects on maintaining

Results and Discussion
Chemistry. The GP inhibitor PSN-357 was obtained according to the method previously described, with some modifications, as outlined in Fig. 2 18 . Commercially available 2-chloro-4-methyl-5-nitropyridine (10) was reacted with diethyl oxalate using potassium ethoxide as the base to give the corresponding derivative 11. Derivative 11 was reduced with iron in saturated aqueous NH 4 Cl followed by cyclization to give pyrrolopyridine ester 12. Hydrolyzed of 12 with NaOH afforded the carboxylic acid derivative 13 in excellent yield. On the other hand, for segment coupling, (S)-N-Boc-4-fluorophenylalanine (14) coupled to the 4-hydroxypiperidine in the presence of HATU and DIPEA to give 15 in 98% yield. Deprotection of the N-Boc group from 15 with HCl gave the amine hydrochloride salt 16. Reaction of 16 with the carboxylic acid derivative 13 in the presence of HATU and DIPEA furnished PSN-357 in 69% yield (Fig. 2).
The synthesis of cholate conjugate 1 and 2 are shown in Fig. 3. Reaction of cholic acid (17) with glycine methyl ester provided amide derivative 18 in 63% yield. Hydrolyzed of 18 with LiOH afforded the glycine conjugate 19 in 76% yield. Compound 19 was treated with PSN-357 in the presence of DCC and DMAP to obtain conjugate 1. In the same fashion, conjugate 2 was prepared from cholic acid and sarcosine methyl ester.
The synthesis of conjugates 3-6 was carried out starting from PSN-357 ( As shown in Fig. 5, the preparation of conjugate 7 via a multistep process by modifying the well-known synthesis method reported in the literature 19 . Coupling of N-Boc-L-Val-OH with L-alanine methyl ester hydrochloride yielded N-protected dipeptide ester 31. Hydrolyzed of 31 with LiOH provided Boc-val-ala-OH 32. The dipeptide derivative 32 was treated with PSN-357 to obtain compound 33 in 52% yield. Subsequent deprotection of compound 33 using TFA gave compound 34, which was reacted with cholic acid through the action of HATU and Et 3 N to give the conjugate 7. Conjugate 8 was obtained in a similar manner using dipeptide linkage prepared from the protected lysine ester (Fig. 6). Treatment of N6-Cbz-L-lysine methyl ester 35 with Boc-L-phenylalanine in the presence of EDCI in DMF at room temperature afforded dipeptide ester 36. The complete synthesis of conjugate 9 containing an aromatic azo-linkage is depicted in Fig. 7. Reduction of o-nitrocinnamic acid 43 under H 2 atmosphere with Pd/C in aqueous NaOH gave the amine intermediate 44.
It was necessary to perform the reduction under basic condition in order to inhibit cyclization of itself.  Enzyme Assay and SAR Analysis. All the compounds were evaluated for their inhibitory activity against rabbit muscle GPa (RMGPa). The activity of RMGPa was measured through detecting the release of phosphate from glucose-1-phosphate in the direction of glycogen synthesis 20 . CP-91149, a well-known allosteric GP inhibitor, which shares the same binding site with PSN-357, was used as the positive control and the results are summarized in Table 1 21 . The assay results showed that most of the synthesized compounds exhibited moderate to good inhibitory activities against RMGPa. Compared to PSN-357 (IC 50 = 0.42 μ M), introduction of cholic acid group to PSN-357 resulted in a great loss of activity (1-3, 5-9) or no activity (4). This remarkable loss of activity was probably due to the direct steric interference by such bulky substituent of cholic acid group. Among the compounds, conjugate 1 (IC 50 = 5.9 μ M) displayed the most efficient inhibition. While the GP inhibition of the intermediates (e.g. 25-28, 33, 40, 42, 48) seemed much acceptable, indicating that introduction of different substitutions in small size on the hydroxyl moiety of PSN-357 resulted in a slight loss of potency. The results is consistent with the SARs of PSN-357 that the hydroxyl of piperidine is not an essential group for the GP inhibitory activity that could be modified slightly 9 . In general, the L-serine derivative 28 (IC 50 = 0.52 μ M) was the most active of the series, being approximately 1.25-fold less potent than that of PSN-357.

Cell Assay and SAR Analysis.
To evaluate the effects of all compounds in cells, the glycogenolysis assays were established in both rat and human liver cells based on the published method 20 . These results are summarized in Table 2. Two of the conjugates revealed excellent inhibitory activity in the cellular assays. Of these, conjugate 9 showed the best activity in isolated rat hepatocytes and HepG2 cells, with IC 50 value of 12.3 μ M and 6.4 μ M, respectively. Likewise, conjugate 6 showed IC 50 value of 13.4 μ M in isolated rat hepatocytes and 6.1 μ M in HepG2 cells, respectively. The inhibitory activities of the derivatives of PSN-357 (e.g. 25-28) were also explored. It is not surprising that the derivatives still retained micromolar inhibitory activities. Introduction of steric bulks to the hydroxyl group (e.g. 33, 40, 42, 48) led to a complete loss of activity. Data analysis indicated no clear SAR for the substitutents in the cell-based assays.
In Vitro Stability studies. All the conjugates were tested for their chemical and metabolic stability in multiple assays, including simulated gastric fluid (SGF), simulated interstinal fluid (SIF), mouse plasma, and mouse liver microsome (MLM).
For the stability in SGF (Fig. 8) and SIF (Fig. 9), the conjugates could be divided into three groups: (1) stable conjugates 2, 6 and 9. All of them were stable in SGF for 24 h with almost no detectable PSN-357, but in SIF, conjugate 6, being stable up to 24 h, were much more stable than 2 and 9. Still, 2 and 9 were relatively stable than other conjugates that no more than 20% of the two compounds were degraded during the 24 h incubation in SIF; (2) unstable conjugates 1 and 7. The two conjugates were degraded within 6 h and 1 h in SGF and SIF, respectively, with alomost no conjugates at the end of the 24 h incubation in both SGF and SIF; (3) complicated conjugates 3, 4, 5 and 8. They were relatively stable in SGF over 24 h of incubation, but degraded rapidly in SIF within 1 h. As shown in Fig. 10, PSN-357 was relatively stable in mouse plasma within the 120-min test, while the conjugates degraded and declined in a mono-exponential model, except conjugates 4 and 6. Conjugates 4 and 6 exhibited considerable stablity in mouse plasma as PSN-357 with approximately 4% and 12% degradation, respectively. Additionally, the compounds' stability in microsome were evaluated by measuring the rate of compounds consumpation in MLM and the results are shown in Table 3. PSN-357 demonstrated good metabolic stability in MLM with longer half life (t 1/2 > 145 min) and slower elimination rate (CL int < 9.6 μ L/min/mg protein, CL < 38.0 μ L/min/mg protein). In addition, conjugate 7, it metabolized fastest in MLM, with a t 1/2 of 4.7 min and displayed very high intrinsic hepatic clearance (CL int of 297.4 μ l/min/mg), suggesting the potential for unacceptably high hepatic clearance. Those results indicate that the release of PSN-357 from the conjugates were greatly affecting by the linkers.

Biodistribution and Pharmacokinetic Studies for Compound PSN-357 and Conjugate 6 in Mice.
Based on the results of the potency and in vitro stability studies, conjugate 6 was selected for in vivo pharmacokinetic analysis following a single intravenous injection of 5 mg/kg in male C57 BL/6. Doses of PSN-357 intravenous were used as standard regimens for comparison. The results are shown in Table 4 and Fig. 11. In plasma, the concentration of PSN-357 reached the C max of 628 ng/mL at 5 min and then sharply decreased during 30 min after dosing, but it gradually increased to reach a secondary peak at 60 min perhaps due to enterohepatic circulation, and the value of AUC 0-t is 965 ng/mL.h. For conjugate 6, the concentration reached the C max of 726 ng/mL at 5 min and then rapidly decreased to 97 ng/mL at 240 min after dosing. In addition, the concentration of major metabolite PSN-357 released from 6 reached the C max of 98 ng/mL at 120 min with the AUC 0-t of 691.67 ng/mL.h, about 6 and 1.4-fold lower than that of PSN-357, respectively. On the other hand, in livers, conjugate 6 did not show the highest concentration at 5 min after administration as compared with PSN-357. The concentration of 6 is dramatically increased to reaching a mean C max of 3094.55 ng/mL at about 15 min after dosing, suggesting the redistribution from plasma or other tissues to livers may have happened, then 6 was eliminated during 30-240 min. For PSN-357 released from conjugate 6, the value of C max is 1023 ng/mL, about 6-fold lower than that of PSN-357, but the value of AUC 0-t is 12960 ng/mL.h, about 1.4-fold higher than that of PSN-357 (9711 ng/mL.h), further, (AUC liver /AUC plasma ) of PSN-357 metaboliting from 6 is 18.74, about 2-fold higher than that of PSN-357 (10.06). Those results suggested that conjugate 6 exhibited some targeting effect to liver that might enrich and display a longer duration of action in liver in comparison with PSN-357.
In Vivo Efficacy of Compound PSN-357 and Conjugate 6. In vivo efficacy of compound PSN-357 and conjugate 6 were studied on leptin-deficient ob/ob mice. Metformin was chosen as a positive control. Figure 12 showed that treatment with PSN-357 (50 mg/kg) could significantly reduc the blood glucose (BG) to a nadir of 6.36 ± 1.46 mg/dl at 1 h min vs 12.80 ± 1.58 in control ob/ob mice (p < 0.005), with significant effects also being evident at 2 h (p < 0.005) and 3 h (p < 0.005) vs controls. A similar but somewhat weaker hypoglycemic effect was observed for the conjugate 6 under the same dosage. Glucose lowering was statistically significant at 1 (p < 0.01), 2 (p < 0.005), 3 (p < 0.005), 6 (p < 0.005) and 24 h (p < 0.05), with the largest drop (BG level is around 7.79 mmol/L) occurring at 3 h. Especially, there was still significant decrease in BG levels by conjugate 6 up to 6 h after administration. The effect might be due to the fact that 6 acts in a sustained release and longer acting manners. It is noteworthy that the findings are highly consistent with the results from in vivo pharmacokinetic studies.

Conclusions
In summary, though a strategy of bile acid conjugation, it has been found possible to prepare liver-selective conjugates of PSN-357. The in vitro biological and stability studies of these conjugates were evaluated to supporting the selection of a conjugate candidate for in vivo pharmacokinetics and pharmacological evaluation. Among the conjugates, conjugate 6 exhibited moderate enzyme inhibitory activity, suitable cellular activity and acceptable stability in various biological fluids. This compound is preferentially distributed into liver and possesses a longer    Table 1. RMGPa inhibition assay for conjugates 1-9 and some intermediates. a Each value represents the mean ± S.D. of three determinations. b NI means no inhibition. c CP-91149 was used as positive control.
duration of action than PSN-357 at the same dose. Moreover, conjugate 6 was able to maintain acceptable antidiabetic effects relative to PSN-357. These results implied that the development of liver-selective conjugates might offer a potential opportunity to overcome the muscles side-effects caused by sequence homology of three GP isoforms.

Chemistry section. (The detailed information is in Supplementary information).
Enzyme Kinetics. The inhibitory activity of the test compounds against rabbit muscle glycogen phosphorylase a (GPa) was monitored using microplate reader (BIO-RAD) based on the published method 20 .
Compound IC 50 a (μM, rat hepatocytes) IC 50 a (μM, HepG2 cells) Compound IC 50 a (μM, rat hepatocytes) IC 50 a (μM, HepG2 cells)  Table 2. Glycogenolysis inhibition assay for conjugates 1-9 and some intermediates in liver cells. a Each value represents the mean ± S.D. of three determinations. b NI means no inhibition. c CP-91149 was used as positive control.  In brief, GPa activity was measured in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate. Each test compound was dissolved in DMSO and diluted at different concentrations for IC 50 determination. The enzyme was added into 100 μ L of buffer containing 50 mM Hepes (pH = 7.2), 100 mM KCl, 2.5 mM MgCl 2 , 0.5 mM glucose-1-phosphate, 1 mg/mL glycogen and the test compound in 96-well microplates (Costar). After the addition of 150 μ L of 1 M HCl containing 10 mg/mL ammonium molybdate and 0.38 mg/mL malachite green, reactions were run at 22 °C for 25 min, and then the phosphate absorbance was measured at 655 nm. The IC 50 values were estimated by fitting the inhibition data to a dose-dependent curve using a logistic derivative equation.

Glycogenolysis Inhibition in Rat Hepatocytes and HepG2 cells.
The inhibition of hepatic glycogenolysis was monitored by the measurement of liver glycogen, which was done quantitatively by the anthrone reagent (Sigma) colorimetric method based on the published method 20 . Isolated rat hepatocytes or HepG2 cells (Sigma) were treated with the test compound or DMSO solvent (final concentration, 0.10%), followed by 60-min incubation with 0.3 nM glucagon (GGN). Assays were terminated by centrifugation, and cells were digested with 30% KOH followed by glycogen determination. The IC 50 values were estimated by fitting the inhibition data to a dose-dependent curve using a logistic derivative equation.     2, 6, 24 h) were removed at the end of incubation time and immediately mixed with 800 μ L of cold acetonitrile containing 500 ng/mL tolbutamide (internal standard). Samples were subjected to centrifuge at 4000 rpm, 4 °C for 20 min. Aliquots 60 μ L of supernatant was combined with 120 μ L water for LC-MS/MS analysis.

Stability in Plasma.
Frozen plasma from male C57 BL/6 mice was incubated for 5 min at 37 °C before the addition of the test compound. Then prepared 2 μ M incubation sample, and aliquots of the incubation mixtures (100 μ L) were taken at predetermined time points (10, 30, 60, 120 min) at 37 °C. After incubation, the mixtures was added 400 μ L of 50% ACN/50% MeOH containing internal standard (IS, 200 ng/mL Tolbutamide and 20 ng/mL Buspirone) to each sample tube and then centrifuged at 13000 rpm for 8 min. Blank incubations in the absence of the test compound were also performed. The analyses of test compounds were performed by LC-MS/MS analysis.
Stability in Liver Microsomes. The microsomal pellet was suspended in potassium phosphate buffer (100 mM, pH 7.4), 10 μ M test compound or positive control (diclofenac) was added. Liver microsomes from C57BL/6 mice (BD Gentest) were pooled. The mixture of microsome solution and compound was incubated at 37 °C for about 10 min in the presence of a NADPH-regenerating system, consisting of 0.02 M DL-isocitric acid (trisodium salt), 0.1 mg/mL isocitrate dehydrogenase and 1 mM NADPH. The addition of ice-cold CH 3 CN (including 100 ng/mL tolbutamide as internal standard) terminated the reaction. The mixture was vortexed for approx (30 s), centrifuged (20 min, 4000 rpm) and the supernatant was collected and analysed by LC-MS/MS method.