Design, Synthesis, and Biological Activity of Novel Dicoumarol

Dec 5, 2013 - Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China. J. Me...
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Design, Synthesis, and Biological Activity of Novel Dicoumarol Glucagon-like Peptide 1 Conjugates Jing Han,† Lidan Sun,† Yingying Chu, Zheng Li, Dandan Huang, Xiaoyun Zhu, Hai Qian,* and Wenlong Huang* Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China S Supporting Information *

ABSTRACT: Twelve novel dicoumarol glucagon-like peptide 1 (GLP-1) conjugates were designed, synthesized, and tested for biological activity. All derivatives retained receptor activation efficacy, and exhibited improved albumin affinity and in vitro stability in rat plasma. The in vivo elimination half-lives of 13c and 13l (22.07 and 18.78 h, respectively) were much longer than those of the GLP-1 receptor agonists exendin-4 (2.82 h) and liraglutide (12.53 h). The prolonged in vivo antidiabetic effects of 13c and 13l on db/db mice were confirmed by the hypoglycemic efficacy test and the multiple intraperitoneal glucose tolerance test. Importantly, a once daily administration of 13c to db/db mice for 7 weeks provided long-term beneficial effects by lowering glycated hemoglobin (HbA1c) levels to 5.05%, which was lower than with liraglutide treatment (5.41%). These results suggest that 13c is a promising long-lasting GLP-1 mimetic that may be suitable for clinical use following further research.



by the need to inject twice daily.15 This approach does illustrate, however, the clinical potential of GLP-1 analogues. Other approaches attempted include inhibiting rapid renal clearance through increasing molecular size via polyethylene glycol (PEG) conjugation or by promoting physical and chemical interactions with biological macromolecules or biodegradable polymer microspheres.16−21 These approaches have promoted a number of GLP-1 analogs reaching various stages of the drug development and clinical trials processes.22−25 The use of chemical modification to improve covalent or noncovalent interactions with serum albumin was an effective strategy for prolonging the half-lives of peptide analogues.26,27 D -Ala8, Lys37[2-[2-[2-maleimidopropionamido(ethoxy)ethoxy]acetamide-GLP-1(7−37) (CJC1131), a GLP-1 analogue with a reactive maleimide group at the carboxyl terminus, binded covalently to serum albumin following injection.28 Unfortunately, it exhibited negative side effects when administered to humans, and the development has ceased.29 Acylation (attachment of a fatty acid side chain) facilitated noncovalent binding to serum albumin in the GLP-1 analogue liraglutide.30 This delayed both the absorption and clearance rates of the compound.31 Additionally, a liraglutide analogue lacking a γ-glutamyl linker at the acylation site still possesses enhanced biological function in vitro and protracted antihyperglycaemic effects in vivo.32 GLP-1 analogues containing cysteine attached to various fatty acid side chains proved to be

INTRODUCTION Total diabetes cases are set to increase greatly to 366 million in 2030, with 90−95% type 2 diabetes mellitus (T2DM) cases.1−3 T2DM is a progressive syndrome characterized by the gradual deterioration of β cell function over time.4 To date, many conventional drugs have been widely utilized for diabetes treatments. However, these antidiabetic agents in particular have many drawbacks; thereby more research is still essential for continued improvement diabetic therapies.5 The external administration of GLP-1 receptor agonists is viewed as an effective approach and has been explored intensively.6 GLP-1 belongs to the incretin class of peptide hormones and is produced by the proteolytic processing of preproglucagon with the predominant form in human plasma being GLP-1(7−36)NH2.7 GLP-1 stabilizes and returns to normal postprandial blood glucose levels by suppressing glucagon secretion and stimulating insulin secretion in the short term and by inhibiting β cell apoptosis and stimulating pancreatic β cell proliferation in the longer term.8,9 GLP-1 treatment is favored over insulin because it acts in a glucose-dependent manner that could prevent hypoglycemia shock.10 However, because of rapid enzymatic degradation and rapid renal removal, the short lifespan in vivo (t1/2 ≈ 2 min) of GLP-1 restricts the effectiveness as a type 2 antidiabetic agent.11 Thus, a number of pharmacological strategies have been adopted in order to overcome those limitations.12,13 Synthesis and evaluation of GLP-1 analogues that act as GLP-1 receptor agonists and that possess DPP IV resistant properties led to the development of exendin-4.14 Unfortunately, the therapeutic effectiveness of exendin-4 is diminished © 2013 American Chemical Society

Received: August 2, 2013 Published: December 5, 2013 9955

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Scheme 1. General Synthetic Route of Dicoumarol Maleimidesa

a

Reagents and conditions: (a) glacial acetic acid, reflux; (b) ethanol, reflux; (c) Pd/C, H2, rt; (d) DIC, HOBt, DMAP.

Scheme 2. General Synthetic Route of Dicoumarol GLP-1 Conjugates

sized and covalently coupled with GLP-1 to afford 12 dicoumarol GLP-1 derivatives (13a−l). The structural properties, in vitro biological activity, and physicochemical characteristics of 13a−l were explored. Compounds 13c and 13l were identified as long-acting and highly biologically active GLP-1 derivatives, and their pharmacokinetic and antidiabetic properties were investigated. Anticoagulant effects in SD rats and long-term beneficial effects in db/db mice were also evaluated.

effective, although serum albumin binding requires further improvement.33 In this study, we developed long-acting GLP-1 analogues using site-specific conjugation with dicoumarol to promote noncovalent interactions. Dicoumarol is an oral anticoagulant that interferes with the metabolism of vitamin K. 4-Hydroxycoumarin is known to bind tightly to human serum albumin (HSA).34 Since diabetic patients are at growing risk of atherothrombosis,35 inclusion of dicoumarol may also help to prevent blood clotting. However, the potential toxicity of dicoumarol has to be considered. Chemical modification of GLP-1 is often accompanied by reduced activity.17 Our previous work found that Cys17-Gly8-GLP-1(7−36)-NH2 (9), Cys26-Gly8-GLP-1(7−36)-NH2 (10), Cys34-Gly8-GLP-1(7− 36)-NH2 (11), and Cys37-Gly8-GLP-1(7−37)-NH2 (12) and their cysteine-maleimide-conjugated adducts retained maximal biological activity.33 In this study, three symmetric 4hydroxycoumarin dicoumarol maleimides (8a−c) were synthe-



RESULTS Synthesis and Characterization of Dicoumarol GLP-1 Conjugates. Four cysteine-containing peptides Cys17-Gly8GLP-1(7−36)-NH2 (9), Cys26-Gly8-GLP-1(7−36)-NH2 (10), Cys34-Gly8-GLP-1(7−36)-NH2 (11), Cys37-Gly8-GLP-1(7− 37)-NH2 (12) were synthesized in accordance with standard solid-phase peptide synthesis procedures. Microwave irradiation was applied in coupling and deprotection steps.36 Peptides were 9956

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Figure 1. Structures of dicoumarol GLP-1 conjugates.

in potency over GLP-1(7−36)-NH2, indicating that conjugation on position 26 with dicoumarol maleimide (8a) could improve receptor activation potency. Chemical conjugation of dicoumarol maleimides (8a−c) to Cys17-Gly8-GLP-1(7−36)NH2 reduced the receptor activation potency of 13a−c between 3- and 5-fold compared with Gly8-GLP-1(7−36)NH2), while the potency of 13a−c was hardly affected, suggesting that cysteine−maleimide conjugations near the Nterminus of GLP-1 are tolerated without diminishing activity. This is important, since the N-terminal portion of the peptide is crucial for mediating the cellular response to GLP-1 signaling.39 The receptor activation potency of 8c conjugates were markedly less potent than 8a and 8b (except for 13a−c), indicating that the longer alkyl chain of dicoumarol maleimides may reduce the receptor activation potency (Figure 2). Stability of the GLP-1 Analogues in Rat Plasma. Twelve conjugates (13a−l) were incubated with plasma taken from SD rats over 72 h and compared with Gly8-GLP-1(7−36)NH2, 9−12, exendin-4, and liraglutide. Solid phase extraction was conduct at each time point, and the extract was analyzed using LC−MS/MS to examine plasma stability.40 Gly8-GLP1(7−36)-NH2 possessed a half-life of ∼0.8 h at 37 °C, while exendin-4 and liraglutide exhibited a nearly 8-fold (t1/2 = 6.2 h) and 24-fold (t1/2 = 17.1 h) longer half-life (Figure 3). All dicoumarol GLP-1 conjugates showed better stability than exendin-4, and most were more stable than liraglutide. The

purified by HPLC and characterized using LC−MS (Supporting Information, Table S1). As shown in Scheme 1, three symmetric dicoumarol maleimides (8a−c) were synthesized relatively easily, since maleimide undergoes highly chemoselective Michael additions with thiol groups at neutral pH. The cysteine-containing peptides (9−12) were reacted with dicoumarol maleimides (8a−c) to give the final compounds (Scheme 2). A total of 12 dicoumarol GLP-1 conjugates (Figure 1) were synthesized and purified by preparative RPHPLC and characterized by both HPLC and LC−MS. Quantitative HPLC analysis revealed that the purities of dicoumarol GLP-1 conjugates were over 95% (Supporting Information, Table S1). Receptor Activation Assay. Cyclic adenosine monophoisphate (cAMP) is the primary effector of GLP-1-induced insulin secretion. After activation of the GLP-1 receptor, an increase of intracellular Ca2+ and cAMP-dependent closure of K−ATP channels leads to glucose-dependent secretion of insulin.37 Therefore, cAMP was chosen to probe the GLP-1 receptor activating properties of our compounds. HEK293 cells overexpressing human GLP-1 receptor were used to determine the receptor activation potency of GLP-1(7−36)-NH2, exendin-4, liraglutide, Gly8-GLP-1(7−36)-NH2, 9−12, and 13a−l.38 Most of the dicoumarol GLP-1 conjugates showed high receptor activation efficacy in this assay (Table 1). In particular, compound 13d showed a remarkable 2-fold increase 9957

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± 1.84 mM at 30 min and was reduced slowly thereafter, while liraglutide, exendin-4, 13c, and 13l dramatically normalized average glucose levels to approximately 8.3, 7.6, 6.9, and 7.8 mM, respectively (Table 2). Moreover, plasma glucose level negatively correlated with plasma insulin concentrations. With treatment of exendin-4, liraglutide, 13c, or 13l, the insulin concentrations in plasma were much higher than that of the control at 15 and 45 min and the time courses were comparable (Supporting Information, Table S7). As expected, the measured glucose levels for these treatments were markedly lower than the control at 15, 30, and 60 min (Figure 5A). After oral administration of 10 g/kg glucose, levels of secreted insulin were dramatically increased to 1180.26 ± 115.23 (exendin-4) and 992.08 ± 110.09 pM (liraglutide) at 15 min, which then decreased to 401.80 ± 53.11 and 425.34 ± 35.45 pM, respectively, at 45 min. For 13c and 13l, secreted insulin peaked at 15 min (1274.72 ± 98.95 and 1052.22 ± 70.77 pM, respectively) and returned to 582.08 ± 51.71 and 524.26 ± 89.45 pM, respectively, at 45 min (Supporting Information, Table S7). 13c and 13l induced insulin levels that were comparable to those in liraglutide treated rats for the entire duration of the experimental (120 min). Furthermore, 13c and 13l showed better insulin secretion promoting ability than exendin-4 and liraglutide (Figure 5B). The glucose-lowering abilities of 13c and 13l were slightly better than liraglutide and comparable with exendin-4 (Figure 5A). In Vivo Pharmacokinetics in SD Rats. To determine the in vivo stability of 13c and 13l, pharmacokinetic data were obtained and compared with exendin-4 and liraglutide controls. Dicoumarol maleimide conjugates possessed enhanced pharmacokinetic profiles (Figure 6, Table 2). After subcutaeous administration, the concentration of exendin-4 in plasma increased quickly and then rapidly declined to initial levels. The peak time was less than 1 h (tmax = 0.8 ± 0.38 h) with a calculated elimination half-life (t1/2) of 2.82 ± 0.21 h. Liraglutide peaked at 1.51 ± 0.38 and declined slowly to initial levels with a t1/2 of 12.54 ± 0.95 h. For 13c and 13l, t1/2 values increased to 22.07 ± 1.10 and 18.78 ± 2.79 h, respectively. Additionally, the area under the curve (AUCinf) values for 13c and 13l were 12.9- and 11.7-fold greater than exendin-4, respectively. Furthermore, the mean residence times (MRTs) of 13c and 13l were 33.80 ± 1.02 and 29.81 ± 2.40 h, respectively. This result suggests that these compounds may meet pharmacokinetic standards for a once-a-day therapeutic agent. Multiple Intraperitoneal Glucose Tolerance Test in db/db Mice. Considering the favorable plasma stability data and pharmacokinetic profiles of 13c and 13l, their capability to maintain normal glucose levels over longer durations was further explored using a multiple intraperitoneal glucose tolerance test on C57BL/6J-m+/+Leprdb (db/db) mice. 13c, 13l, exendin-4, liraglutide (25 nmol/kg) were intraperitoneally administrated 0.5 h before the first glucose challenge. Glucose was given at 0, 3, 6, 9, 12, 15, 18, and 21 h. Blood glucose levels of the saline-treated control group quickly peaked over 30 mM within 0.5 h of each glucose challenge and decreased slowly. The hypoglycemic effect of exendin-4 was stable in 0−5 h but decreased significantly after 6 h and nearly disappeared after 9 h (Figure 7A). Interestingly, liraglutide exerted potent synergistic glucose-lowering efficacy between 3 and 17 h but showed no noticeable effect between 0 and 3 h. 13c and 13l maintained blood glucose levels below 12 mM between 0 and 17 h, the entire duration of the experiment, although the glucose-

Table 1. Human GLP-1 Receptor Activation, Plasma Stability, and Albumin Binding of Cysteine-Containing GLP1 Analogues and Dicoumarol GLP-1 Conjugates compda

potency,b EC50 (pM)

plasma half-life (h)

albumin bindingc (%)

GLP-1(7−36)-NH2 exendin-4 liraglutide Gly8-GLP-1(7−36)-NH2 9 10 11 12 13a 13b 13c 13d 13e 13f 13g 13h 13i 13j 13k 13l

3.3 ± 0.8 1.5 ± 0.2 9.3 ± 0.5 8.2 ± 2.6 6.4 ± 1.5 3.2 ± 0.8 4.9 ± 1.2 3.8 ± 0.5 26.1 ± 6.4 33.6 ± 9.5 36.8 ± 8.5 1.5 ± 1.3 3.4 ± 1.7 21.9 ± 5.6 6.5 ± 2.6 7.3 ± 2.9 18.1 ± 4.5 3.0 ± 1.2 3.2 ± 1.3 24.2 ± 9.7

6.2 17.1 0.8 1.1 0.7 0.6 0.9 13.6 20.7 67.9 7.29 21 21.7 19.3 22.1 38.6 9.7 18.6 55.8

22.3 ± 3.6 91.3 ± 2.3 6.3 ± 1.3 7.8 ± 0.8 5.1 ± 1.2 6.1 ± 1.2 9.2 ± 0.6 69.0 ± 1.1 83.8 ± 2.4 96.4 ± 2.4 57.7 ± 4.2 74.9 ± 1.1 81.3 ± 3.3 72.7 ± 1.4 80.2 ± 6.3 83.2 ± 4.2 66.8 ± 6.4 75.2 ± 2.7 94.0 ± 4.8

a 9, 10, 11, and 12 contain Cys at positions 17, 26, 34, and 37, respectively. 13a, 13b, and 13c have a modified Cys at position 17. 13d, 13e, and 13f at position 26; 13g, 13h, and 13i at position 34; 13j, 13k, and 13l at position 37. bThe functional (cAMP) potency data are the mean ± SD of three individual experiments done in triplicate. c Data are the mean ± SD (n = 4).

half-lives of most of the conjugates were over 9 h, and 13c exhibited the longest half-life of 67.9 h (Figure 3A). Notably, the plasma half-life values of 13a−l correlated well with the alkyl chain length of these dicoumarol malemides, with a halflife of 13.6 h for the C2 alkyl chain (13a), 20.7 h for the C5 chain (13b), and 67.9 h for the C11 chain (13c). 13d−l showed comparable results (Table 1, Figure 4). Binding to Human Serum Albumin. We predicted that the increased albumin binding might lead to the increased metabolic stability, and this was investigated for 13a−l, 9−12, Gly8-GLP-1(7−36)-NH2, exendin-4, and liraglutide. Under physiological conditions, the binding rates of Gly8-GLP-1(7− 36)-NH2, exendin-4, and liraglutide to albumin resin were 6.3 ± 1.3%, 22.3 ± 3.6%, and 91.3 ± 2.3%, respectively (Table 1). With Gly8-GLP-1(7−36)-NH2, exendin-4, and liraglutide used as control, all dicoumarol GLP-1 conjugates exerted improved albumin affinity compared with Gly8-GLP-1(7−36)-NH2, highly increased in the cases of 13c and 13l which were comparable with liraglutide. Albumin binding correlated well with plasma stability (Figure 4). Glucoregulatory and Insulin Secretion Assay in Sprague−Dawley (SD) Rats. The exceptionally high plasma stability and albumin binding ability of 13c and 13l boded well for prolonging activity in vivo. To investigate this, glucoregulatory and insulin secretion assays were conducted. Specifically, the in vivo antidiabetic activities of 13c and 13l were assessed using the oral glucose tolerance test (OGTT) on SD rats.41 Intraperitoneal (ip) administration of 13c, 13l, liraglutide, or exendin-4 (25 nmol/kg) all promoted a remarkable improvement in glucose tolerance. The average blood glucose level of the saline-injected mice peaked to 13.32 9958

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Figure 2. Correlation between the alkyl chain length of dicoumarol maleimides (8a−c) and the receptor activation potency of dicoumarol GLP-1 conjugates (13a−l): n = 3, (∗) p < 0.05.

Preliminary Assessment of Anticoagulant Activity in Male SD Rats. To determine if 13c and 13l possessed anticoagulant activity, SD rats were administrated intravenously with 13c or 13l in two doses for 6 days. On the seventh day, following an overnight fast (12 h), blood samples were collected in microcentrifuge tubes containing sodium citrate before dosing. Anticoagulant activity was measured with a coagulometer (Sysmex CA-7000) according to the manufacturer’s instructions. Contrary to our expectations, 13c and 13l did not prolong the activated prothrombin time (PT), thrombin time (TT), or prothrombin time (APTT) after treatment (Table 3). Chronic in Vivo Studies in db/db Mice. To further explore the in vivo antidiabetic activity of 13c, the chronic effects were also examined in db/db mice. 13c (25/50 nmol/ kg) and liraglutide (25 nmol/kg) were intraperitoneally administrated once daily. Glycated hemoglobin (HbA1c) was measured, as this is a well-known sensitive index of glycemic control which is widely used to indicate cumulative blood glucose concentrations caused by nonenzymatic irreversible glycation.43 The HbA1c values were matched well initially in db/db groups which were significantly higher than that in wildtype nondiabetic C57BL/6 littermates (Figure 9A). During the 7-week repeated dosing period, a gradual reduction of HbA1c was recorded in the diabetic mice daily treated with either 25 nmol/kg or 50 nmol/kg of 13c (ip) whereas HbA1c in the control group remained high through the experiment. After a 5week treatment, HbA1c levels were significantly more reduced in the 13c (25/50 nmol/kg) treated group than the liraglutide group to the extent that HbA1c levels became indistinguishable

lowering effects were slightly reduced after 18 h (Figure 7). Even so, blood glucose levels were still maintained below 16 mM between 18 and 21 h (Figure 7B). Blood glucose levels increased to 13.80 ± 1.56 and 16.25 ± 0.64 mM at 21.50 h in the 13c and 13l groups, respectively, and rapidly decreased thereafter (Figure 7B). This suggests that these compounds still exerted glucoregulatory effects, whereas liraglutide was much less effective between 18 and 24 h. In conclusion, a single 25 nmol/kg dose of 13c or 13l retained glucoregulatory activity for 24 h, which was more long-acting than exendin-4 and liraglutide. Hypoglycemic Duration Test in db/db Mice. The hypoglycemic effects of 13c and 13l were assessed at two doses (25 and 150 nmol/kg, ip) in db/db mice, and a euglycemic duration under 8.35 mM of the blood glucose level was considered an indicator of potential as an antidiabetic treatment.42 As shown in Figure 8, exendin-4 (25 nmol/kg) and liraglutide could normalize blood glucose for 4.8 and 19.0 h after injection while the hypoglycemic durations of 13c and 13l at a dose of 25 nmol/kg were approximately 24.7 and 22.5 h, both of which were nearly 5-fold greater than exendin-4 and much better than liraglutide. Moreover, the times required to return glucose levels below 8.35 mM were ∼24.7 and 22.5 h for 13c and 13l treated mice, respectively, compared with ∼4.8 h for exendin-4 and ∼19.0 h in liraglutide (Figure 8B). The t∼8.35mM values for 13c and 13l in mice administered the higher 150 nmol/kg dose were 35.8 and 30.4 h, respectively. Calculated glucose AUC0−48h values also showed that 13c had better antidiabetic activity than exendin-4 and liraglutide at a dose of 25 nmol/kg (Figure 8C, p < 0.001). 9959

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Figure 3. Degradation of dicoumarol GLP-1 conjugates (13a−l), 9−12, Gly8-GLP-1(7−36)-NH2, exendin-4, and liraglutide. Data are expressed as the mean ± SD, n = 4.

length of the alkyl chain (Table 1, Figure 2). This was probably because the dicoumarol maleimide was attached near the Nterminus, which inhibited the GLP-1 receptor activating ability of 13a−c. In vitro plasma stability and albumin binding experiments showed that these properties were improved in all conjugated compounds (13a−l) but to different degrees (Table 1), and plasma stability was dependent on albumin binding ability (Figure 4). 13c and 13l exhibited the best albumin binding ability and the longest plasma half-life, respectively. As the most promising compounds, these were further examined for glucose-lowering and in vivo insulinotropic activity. Despite reduced receptor activation ability due to dicoumarol maleimide conjugation, 13c and 13l showed comparable glucose-lowering ability and insulinotropic activity to exendin4 and liraglutide (Figure 5). Notably, 13c and 13l were acting via the glucose-dependent mechanism, which suggested that they could be safely used without inducing hypoglycemia. In vivo pharmacokinetic profiles of 13c and 13l were explored, and the improved elimination half-life values may result from enzyme metabolism resistance and reduced renal clearance due to the strong albumin binding. The correlation between albumin binding and pharmacokinetic profile is similar with previous reports on fatty acid modified GLP-1 analogues, which was also attributed to interactions with serum albumin.18,44 IPGTT and hypoglycemic duration tests in db/db mice further confirmed the prolonged glucose-lowering activity of 13c and 13l. Independent of the dose injected, the hypoglycemic durations in db/db mice treated with 13c and 13l were

from those of nondiabetic wild-type mice in 13c treated mice. This result is promising for future clinical studies. Body weight gain was also measured and was suppressed after chronic treatment of either 13c (25/50 nmol/kg) or liraglutide, while this continually increased by ∼14 g in control group and almost remained unchanged in wild-type mice as expected. Furthermore, weight gain was suppressed in a dose-dependent manner in 13c treated mice (Figure 9B). After long-term treatment of 13c, the glucose tolerance improvement was assessed through the IPGTT test on day 0 and day 52. On day 0, the blood glucose profiles of all groups showed no statistical difference (Figure 10). After a 7-week treatment, 13c (25/50 nmol/kg) and liraglutide (25 nmol/kg) treated mice exerted marked reductions in blood glucose levels that returned to baseline 120 min after the glucose challenge. In contrast, saline treated mice exhibited a typical diabetic pattern after the load of glucose (20.6 ± 0.99 mmol/L).



DISCUSSION AND CONCLUSIONS In the present study, a series of novel GLP-1 conjugates were designed and synthesized to develop long-acting incretin mimetics. In GLP-1 receptor activation experiments, the receptor activation potentials of compounds 13d−l correlated with the length of the alkyl chain attached to dicoumarol malemides (8a−c) and were barely affected by the positions of the cysteine residues (Cys26, Cys34, Cys37) used for conjugation. Moreover, the receptor activation abilities of compounds 13a− c, which were less potent than 13d−l, were independent of the 9960

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Figure 4. Metabolic stability in rat plasma. Half life (t1/2) and % binding to human serum albumin are shown.

Table 2. Pharmacokinetic Profiles of Exendin-4, Liraglutide, 13c, and 13l in SD Ratsa compd exendin-4 liraglutide 13c 13l

Tmax (h) 0.80 1.51 1.82 1.30

± ± ± ±

0.38 0.38 0.39 0.34

Cmax (ng/mL)

AUCinf (ng h−1 mL−1)

t1/2 (h)

MRT (h)

± ± ± ±

3153.07 ± 131.25 18073.59 ± 911.64 40580.43 ± 1949.67 36839.11 ± 1629.21

2.82 ± 0.21 12.54 ± 0.95 22.07 ± 1.10 18.78 ± 2.79

4.17 ± 0.10 18.34 ± 1.21 33.80 ± 1.02 29.81 ± 2.40

771.49 901.89 880.07 907.30

71.41 18.56 14.13 31.81

Data are shown as the mean ± SD (n = 6). Tmax, time to reach maximum plasma concentration; Cmax, maximum plasma concentration; AUCinf, area under the curve from zero to infinity; t1/2, elimination half-life; MRT, mean residence time.

a

be explained by similar off-target effects, or perhaps the doses were below the minimum required for anticoagulation activity. The chemical linker attaching the dicoumarol may have an influence and should be investigated. Dicoumarol fragments may also exhibit toxicity as well as anticoagulation, and the acute and chronic toxicity and pharmacokinetic properties are currently under investigation. In summary, cysteine−dicoumarol conjugates are potentially useful long-acting GLP-1 derivatives. In particular, 13c showed preserved biological activity, a prolonged pharmacokinetic profile, long-acting antidiabetic properties, and long-term beneficial effects. This compound is deserving of further investigation to assess its therapeutic potential in T2DM.

significantly greater than exendin-4 and liraglutide. Given that drug clearance in rodents was much faster than in man, the antidiabetic durations may be substantially greater in humans than the 35.8 and 30.4 h observed for 13c and 13l, respectively, in mice. A once daily injection of 13c to db/db mice achieved long-term beneficial effects as shown by lower HbA1c levels and improved glucose tolerance. The long-term treatment effects of 13c were consistent with chronic peripherally administered liraglutide and included weight loss and reduced HbA1c.45 As with native GLP-1, the protracted activity of β cell neogenesis and/or proliferation of 13c presumably enhanced the glucose tolerance in db/db mice.46 Preliminary assessment of the anticoagulant effects of 13c and 13l showed negative results. This was somewhat surprising, given that dicoumarol is a known anticoagulant. This result may 9961

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Figure 5. In vivo biological activity of 13c and 13l: (A) glucose-lowering and insulinotropic activities of exendin-4, liraglutide, 13c, and 13l (25 nmol/kg) determined using OGTT in SD rats; (B) calculated plasma insulin AUC0−180min in exendin-4, liraglutide, 13c, and 13l (25 nmol/kg) treated SD rats. Data are shown as the mean ± SD, n = 6, (∗∗) p < 0.01, (∗∗∗) p < 0.001. Synthesis and HPLC Purification of Compounds 9−12. Peptides 9−12 were prepared and purified using previously described methodology.33 Briefly, Fmoc Rink amide-MBHA resin (0.015 mmol) was deprotected with 25% piperidine in 5 mL of DMF for 4 min under microwave irradiation (microwave power of 10 W). A mixed solution of 0.045 mmol of first amino acid, 0.045 mmol of HBTU, 0.045 mmol of HOBT, and 0.090 mmol of DIPEA dissolved in 4 mL of DMF was added after washing three times with DMF. The mixture was bubbled with N2 for 10 min under microwave irradiation (10 W) and washed three times with DMF. Deprotection and coupling were repeated with the relevant Fmoc-protected amino acids, and peptides were cleaved with 7 mL of reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 1.5 h at room temperature. Then peptides were precipitated by addition of 50 mL of cold ethyl ether. After centrifugation, the crude peptides were purified using a Shimadzu preparative RP-HPLC. Conditions for purification were as follows. Samples were injected into a Shimadzu C18 reverse phase column (5 μm, 340 mm × 28 mm) equilibrated in buffer A (water with 0.1% TFA) and purified using a linear gradient from 30% to 75% buffer B (acetonitrile and 0.1% TFA) over 30 min at a flow rate of 6.0 mL/min with UV detection at 214 nm. The molecular mass of the purified peptides was confirmed by LC−MS. General Procedure I for Preparation of Compounds 3a−c. Compounds 2a−c and a 1.2 mol equiv of furan-2,5-dione in 20 mL of acetic acid were stirred at 120 °C for 6 h. The reaction mixture was poured into water after cooling to room temperature and extracted with ethyl acetate (3 × 20 mL). The organic layers were combined, washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give the crude product. Purification was performed by column chromatography in 1:6 ethyl acetate/petroleum ether (v/v). 3-(2, 5-Dioxo-2H-pyrrol-1(5H)-yl)propanoic Acid (3a). Furan2,5-dione (1) (0.35 g, 3.6 mmol) was reacted with compound 2a (0.27 g, 3 mmol) to furnish 3a as a white solid (0.42 g, 82%); mp 106−109 °C. 1H NMR (DMSO-d6, 300 MHz): δ 12.37 (s, 1H, COOH), 7.03 (s, 2H, COCHCHCO), 3.61 (t, J = 7.1 Hz, 2H, NCH2), 2.49 (t, J = 7.4 Hz, 2H, CH2COOH). ESI-MS: m/z calculated for C7H7NO4 [M+] 169.0; found 168.0. 6-(2,5-Dioxo-2H-pyrrol-1(5H)-yl)hexanoic Acid (3b). Furan2,5-dione (1) (0.35 g, 3.6 mmol) was reacted with compound 2b (0.39 g, 3 mmol) to furnish 3b as a white solid (0.47 g, 74%); mp 90−91 °C. 1 H NMR (DMSO-d6, 300 MHz): δ 11.96 (s, 1H, COOH), 6.69 (s, 2H, COCHCHCO), 3.52 (t, 2H, J = 7.2 Hz, NCH2CH2), 2.35 (t, 2H, J = 7.4 Hz, CH2COOH), 1.72−1.57 (m, 4H, NCH2CH2CH2CH2), 1.39−1.25 (m, 2H, NCH2CH2CH2CH2). ESIMS: m/z calculated for C10H13NO4 [M+] 211.1; found 210.2.

Figure 6. Pharmacokinetic properties of exendin-4, liraglutide, 13c, and 13l in SD rats after subcutaneous injection (15 nmol/kg, n = 6). Data are shown as the mean ± SD.



EXPERIMENTAL SECTION

Materials and Animals. Reagents and materials were purchased from the following companies: Fmoc Rink amide-MBHA resin and Fmoc-protected amino acids, GL Biochem (Shanghai, China); HPLC grade acetonitrile and methanol, Merck (Darmstadt, Germany); liraglutide, exendin-4, and Gly8-GLP-1(7−36)-NH2, GL Biochem (Shanghai, China); cAMP dynamic kit, Cisbio (Bedford, MA); rat insulin ELISA kit, Millipore (Billerica, MA); HbA1c kit, Glycosal (Deeside, UK). All other reagents, unless otherwise indicated, were obtained from Sigma-Aldrich Co. (Saint Louis, MO) and used as received. Microwave irradiation procedures were performed in a Discover focused single mode microwave synthesis system (CEM, NC), which produced continuous irradiation at 2450 MHz. HPLC analysis and purification were performed on a Shimadzu 2010C HPLC system and a Shimadzu LC-10 preparative RP-HPLC system, respectively. ESI mass spectra were obtained with a Waters ACQUITY UPLC system (Milford, MA). Sprague−Dawley (SD) rats (male, 200−250 g) were purchased from the Comparative Medical Center of Yangzhou University (Jiangsu, China). C57BL/6J-m+/+Leprdb (db/ db) mice and wild-type nondiabetic C57BL/6 littermates (male and female, 6−8 weeks old) were obtained from the Model Animal Research Center of Nanjing University (Jiangsu, China). All animal experimental protocols adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 85-23, revised 1986). 9962

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Figure 7. Longer term (24 h) glucose-lowering effects of 13c and 13l studied by the intraperitoneal glucose tolerance test in db/db mice. Plasma glucose levels are shown in panels A and B. Glucose loading times are indicated by arrows. Data are shown as the ± SD, n = 6. 12-(2,5-Dioxo-2H-pyrrol-1(5H)-yl)dodecanoic Acid (3c). Furan-2, 5-dione (1) (0.35 g, 3.6 mmol) reacted with compound 2c (0.65 g, 3 mmol) to furnish 3c as a white solid (0.69 g, 78%); mp 90− 92 °C. 1H NMR (CDCl3, 300 MHz): δ 11.93 (s, 1H, COOH), 7.00 (s, 2H, COCHCHCO), 3.37 (t, 2H, J = 7.0 Hz, NCH2), 2.18 (t, 2H, J = 7.3 Hz, CH2COOH), 1.49−1.45 (m, 4H, NCH2CH2(CH2)7CH2), 1.22 (s, 14H, NCH2CH2(CH2)7CH2). ESI-MS: m/z calculated for C16H25NO4 [M+] 295.2; found 294.1. 4-Hydroxy-3-((4-hydroxy-2-oxochroman-3-yl)(4nitrophenyl)methyl)-2H-chromen-2-one (6). 4-Nitrobenzaldehyde (5) (0.30 g, 2 mmol) and 4-hydroxy-2H-chromen-2-one (4) (0.65 g, 4 mmol) in 15 mL of EtOH were stirred at 80 °C for 12 h. The resulting precipitate was collected by filtration, washed with EtOH (3 × reaction volume), and dried overnight at 90 °C (0.79 g, 86%); mp 241−243 °C. 1H NMR (DMSO-d6, 300 MHz): δ 8.08 (d, J = 7.8 Hz, 2H, Ar-H), 7.84 (d, J = 7.8 Hz, 2H, Ar-H), 7.58−7.53 (m, 2H, Ar-

H), 7.40−7.25 (m, 6H, Ar-H), 6.36 (s, 1H, CH). ESI-MS: m/z calculated for C25H15NO8 [M+] 457.1; found 456.0. 3-((4-Aminophenyl)(4-hydroxy-2-oxochroman-3-yl)methyl)4-hydroxy-2H-chromen-2-one (7). A solution of 6 (0.46 g, 1 mmol) in THF (15 mL) was subjected to hydrogenation under 40 psi of H2 in the presence of 5% Pd/C catalyst (0.26 g) for 24 h. Following filtration, the filtrate was concentrated in vacuo and the crude material was purified by column chromatography in 10:1 dichloromethane/ methanol (v/v); mp 200−203 °C. 1H NMR (DMSO-d6, 300 MHz): δ 7.79 (d, J = 7.8 Hz, 2H, Ar-H), 7.51 (t, J = 7.7 Hz, 2H, Ar-H), 7.28− 7.13 (m, 8H, Ar-H), 6.26 (s, 1H, CH), 3.39 (s, 2H, NH2). ESI-MS: m/ z calculated for C25H17NO6 [M+] 427.1; found 426.0. General Procedure II for the Preparation of Compounds 8a−c. HOBt (1.2 M equiv) and compounds 3a−c (1.0 M equiv) were dissolved in dry THF (5 mmol/mL). DIC (1.1 M equiv) was added. The solution was stirred for 30 min and transferred to a second flask, where a solution of 7 (1.0 M equiv) and DMAP (0.1 M equiv) in THF 9963

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Figure 8. Hypoglycemic effects of 13c and 13l in db/db mice: (A) time course average blood glucose levels of db/db mice after ip injection of exendin-4 (25 nmol/kg), liraglutide (25 nmol/kg), 13c (25/150 nmol/kg), and 13l (25/150 nmol/kg); (B) time taken to return from hypoglycemic to normal (8.35 mM); (C) hypoglycemic effects of exendin-4 (25 nmol/kg), liraglutide (25 nmol/kg), 13c (25/150 nmol/kg), and 13l (25/150 nmol/kg) based on calculated glucose AUC0−48h values. Data are shown as the mean ± SD, n = 6, (∗∗∗) p < 0.001 compared with exendin-4 treatment, and (▲▲) p < 0.01 and (▲▲▲) p < 0.001 compared with liraglutide treatment. MHz): δ 9.85 (s, 1H, CONH), 7.81 (d, J = 7.7 Hz, 2H, Ar-H), 7.50 (t, J = 7.6 Hz, 2H, Ar-H), 7.30−7.20 (m, 6H, Ar-H), 7.01 (s, 2H, Ar-H), 6.96 (s, 2H, COCHCHCO), 6.19 (s, 1H, CH), 3.68 (t, J = 7.1 Hz, 2H, NCH2), 3.17 (s, 2H, COCH2). ESI-MS: m/z calculated for C32H22N2O9 [M +] 578.1; found 577.6. 6-(2,5-Dioxo-2H-pyrrol-1(5H)-yl)-N-(4-((4-hydroxy-2-oxo-2Hchromen-3-yl)(4-hydroxy-2-oxochroman-3-yl)methyl)phenyl)hexanamide (8b). 3-((4-Aminophenyl)(4-hydroxy-2-oxochroman-3yl)methyl)-4-hydroxy-2H-chromen-2-one (7) (0.43 g, 1 mmol) was reacted with compound 3b (0.21 g, 1 mmol) to furnish 8b as a red solid (0.50 g, 81%); mp 232−234 °C. 1H NMR (DMSO-d6, 300 MHz): δ 9.70 (s, 1H, NH), 7.81 (d, J = 7.6 Hz, 2H, Ar-H), 7.51 (t, J = 7.3 Hz, 2H, Ar-H), 7.35−7.21 (m, 6H, Ar-H), 6.98 (s, 4H, Ar-H, COCHCHCO), 3.48 (t, J = 6.9 Hz, 2H, NCH2), 2.22 (t, J = 6.9 Hz, 2H,COCH2), 1.55−1.47 (m, 4H, NCH2CH2CH2CH2), 1.24 (s, 2H, NCH2CH2CH2CH2). ESI-MS: m/z calculated for C35H28N2O9 [M+] 620.2; found 619.0. 12-(2,5-Dioxo-2H-pyrrol-1(5H)-yl)-N-(4-((4-hydroxy-2-oxo2H-chromen-3-yl)(4-hydroxy-2-oxochroman-3-yl)methyl)phenyl)dodecanamide (8c). 3-((4-Aminophenyl)(4-hydroxy-2-oxochroman-3-yl)methyl)-4-hydroxy-2H-chromen-2-one (7) (0.42 g, 1 mmol) was reacted with compound 3c (0.30 g, 1 mmol) to furnish 8c as a red solid (0.50 g, 71%); mp 203−205 °C. 1H NMR (DMSO-d6, 300 MHz): δ 9.74 (s, 1H, CONH), 7.85 (d, J = 7.8 Hz, 2H, Ar-H), 7.55 (t, J = 7.6 Hz, 2H, Ar-H), 7.40−7.25 (m, 6H, Ar-H), 7.03 (s, 2H, Ar-H), 7.00 (s, 2H, COCHCHCO), 6.24 (s, 1H, CH), 3.36 (t, J = 7.0 Hz, 2H, NCH2), 2.24 (t, J = 7.1 Hz, 2H, COCH2), 1.55−1.44 (m,

Table 3. Anticoagulant Activity of Dicoumarin, 13c, and 13l in SD Ratsa sample control 13c (25 nmol/kg) 13c (150 nmol/kg) 13l (25 nmol/kg) 13l (150 nmol/kg) dicoumarin (1.0 μmol/kg) a

PT (s) 13.50 13.39 13.55 13.39 13.53 25.22

± ± ± ± ± ±

0.32 0.31 0.15 0.35 0.26 0.86

TT (s) 19.38 19.63 19.49 19.40 19.80 36.85

± ± ± ± ± ±

0.72 0.63 0.64 0.73 0.69 0.87

APTT (s) 26.50 26.39 26.38 26.76 26.48 56.36

± ± ± ± ± ±

0.81 0.58 0.85 0.77 0.64 0.79

Data are shown as the mean ± SD (n = 6).

(3 mmol/mL) had been prepared. The mixture was stirred overnight at room temperature, then diluted with 20 mL of water and extracted with ethyl acetate (3 × 10 mL). The organic layer was washed with water (10 mL), a saturated solution of K2CO3 (3 × 10 mL), 1 M HCl (3 × 10 mL), and water again (2 × 10 mL) and dried with Na2SO4, and residual solvent was removed under reduced pressure. The crude product was purified by column chromatography with 10:1 dichloromethane/methanol as eluent. 3-(2,5-Dioxo-2H-pyrrol-1(5H)-yl)-N-(4-((4-hydroxy-2-oxo-2Hchromen-3-yl)(4-hydroxy-2-oxochroman-3-yl)methyl)phenyl)propanamides (8a). 3-((4-Aminophenyl)(4-hydroxy-2-oxochroman3-yl)methyl)-4-hydroxy-2H-chromen-2-one (7) (0.43 g, 1 mmol) was reacted with compound 3a (0.17 g, 1 mmol) to furnish 8a as a red solid (0.68 g, 85%); mp 312−314 °C. 1H NMR (DMSO-d6, 300 9964

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Figure 9. Chronic effects of 13c and liraglutide in db/db mice: (A) HbA1c measured weekly; (B) body weight gain. Data are shown as the mean ± SD, n = 8.

Figure 10. Intraperitoneal glucose tolerance test in diabetic db/db mice. 13c and liraglutide were chronically administered on day 0 and day 52. Data are shown as the mean ± SD, n = 8. 4H, NCH2CH2(CH2)7CH2), 1.22 (s, 14H, NCH2CH2(CH2)7CH2). ESI-MS: m/z calculated for C41H40N2O9 [M+] 704.3; found 703.1. General Synthetic Route of Dicoumarol GLP-1 Conjugates (13a−l). Cysteine modified peptides (9−12, 5 μmol) were conjugated with dicoumarol maleimides (8a−c, 12 μmol) in 5 mL of 0.05 M sodium phosphate buffer, pH 7.0, as previous reported.33 The reaction mixture was stirred at 20 °C under N2 for 1.5 h until HPLC confirmed completion using the following conditions: a C18 column (5 μm, 150 mm × 4.6 mm) equilibrated in buffer A (water, 0.1% TFA), elution with a linear gradient of 20−80% buffer B (acetonitrile with 0.1% TFA) over 20 min at a flow rate of 1 mL/min, and UV detection at 214 nm. Crude conjugates were purified on a Shimadzu preparative RP-HPLC system using a Shimadzu C18 reverse phase column (5 μm, 340 mm × 28 mm) equilibrated in buffer A (water, 0.1% TFA) and eluted with a linear gradient of 40−90% buffer B (acetonitrile, 0.1% TFA) over 30 min at a flow rate of 6.0 mL/min with UV detection at 214 nm. The molecular mass of the purified conjugates were confirmed by LC-MS. Purity Characterizations of Dicoumarol GLP-1 Conjugates. A linear gradient elution was set up to evaluate the purity of all tested

compounds (13a−l) used for subsequent biological assays. The HPLC column used was a Shimadzu C18 reversed-phase column (5 μm, 150 mm × 4.6 mm). Mobile phases consisted of water and acetonitrile, both added with trifluoroacetic acid (TFA) at 0.1% v/v and at a flow rate of 1 mL min−1. Conditions chosen were the following: eluent A, acetonitrile + 0.1% TFA; eluent B, water + 0.1% TFA. T(0 min): 20% A: 80% B. T(20 min): 80% A/20% B returning to initial conditions after 0.5 min, followed by 5 min reequilibration time. The wavelength was 214 nm, and the injection volume was 10 μL. Human GLP-1 Receptor Activation Measurement Using the cAMP Assay. HEK293 cells stably expressing human GLP-1 receptor were used for measurement of intracellular cAMP using a slightly modification of a previously described method.33 Cells were grown at 37 °C in 5% CO2 in Dulbecco’s modified Eagle medium-31053 (Invitrogen, CA) supplemented with 0.5% fetal bovine serum (FBS), 2 mM glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin, and 20 mM HEPES. Two hour before testing, cells were resuspended in the aforementioned medium and plated in 96-well half area solid black microplates. Compounds were solubilized in DMSO, diluted in medium containing 0.1% bovine serum albumin (BSA) with fraction V 9965

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substituted for 0.5% FBS, and added to cells. Following a 20 min incubation, cells were assayed for cAMP using the Cisbio cAMP dynamic 2 kit using homogeneous time-resolved fluorescence technology (Bedford, MA). Fluorescence was measured according to the manufacturer’s instructions using an Envision 2,104 multilabel reader (PerkinElmer, U.K.). The potency of the conjugates (EC50 values) was determined by sigmoidal curve fitting using GraphPad Prism version 5.0 (San Diego, CA). Compound Stability in Rat Plasma. The stability of these conjugates was assessed in rat plasma collected from adult male SD rats, as previously described.33 The plasma was stored at −20 °C until needed. In vitro stability of 13a−l, 9−12, exendin-4, liraglutide, and Gly8-GLP-1(7−36)-NH2 was measured using an initial concentration of 1000 ng/mL of each peptide in rat plasma at 37 °C. An amount of 100 μL of plasma was removed from the incubations at 0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72, and 92 h time points and subjected to solid phase extraction on a Waters Oasis HLB 96-well plate (Milford, MA). An amount of 20 μL of extract was injected onto the LC−MS/MS system. Peptides were detected by multiple reaction monitoring (MRM) using electrospray ionization mass spectrometry (ESI-MS) on an Applied Biosystems Sciex API-4000 instrument (Foster City, CA) fitted with a TurboIonSpray source. RPLC separation was performed on a Waters Acquity UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm) equilibrated in buffer A (water, 0.2% formic acid) and elution was with a linear gradient of 5−95% buffer B (acetonitrile, 0.2% formic acid) over 4 min at a flow rate of 0.3 mL/min. Human Serum Albumin Binding Assay. As previously described, the albumin binding characteristics of 13a−l, 9−12, exendin-4, liraglutide, and Gly8-GLP-1(7−36)-NH2 were investigated using an albumin binding assay with albumin-conjugated Sepharose resin according to the manufacturer’s instructions.33,47 An amount of 50 μL of 100 μg/mL Gly8-GLP-1(7−36)-NH2, exendin-4, liraglutide, 9−12, or 13a−l in PBS was mixed with HSA resin and incubated for 3 h at room temperature (25 °C). The resin and supernatant were separated by centrifugation (1000 rpm, 10 min), and unbound peptides were determined using a PIERCE Mico BCA protein assay kit (Rockford, IL). Nonspecific absorption of peptides on albumin-free resin was determined using NHS-inactivated resin in a similar manner. Glucoregulatory and Insulin Secretion Assay in Sprague− Dawley (SD) Rats. Glucoregulatory and insulin secretion assays first involved intraperitoneal injection of 13c, 13l, exendin-4, and liraglutide into SD rats (n = 6/group, 200−250 g), as previously described.33 Saline was used in control rats. Briefly, SD rats were fasted overnight (12 h). At 0.5 h prior to oral glucose loading (10 g/kg, the standard dose for oral glucose administration), male SD rats were administered saline, exendin-4, liraglutide, 13c, or 13l (ip, 25 nmol/kg). For the glucoregulatory assay, blood was collected from tail tip veins at −30, 0, 15, 30, 45, 60, 90, 120, and 180 min, and blood glucose was measured with an Ascensia Breeze 2 blood glucose monitor (Bayer, Germany). For the insulin secretion assay, blood samples were assayed using a rat insulin ELISA kit. In Vivo Pharmacokinetics in SD Rats. The pharmacokinetic parameters of exendin-4, liraglutide, 13c, and 13l were determined in male SD rats (n = 6/group, 200−250 g) using a modification of a previously described method.33 Following an overnight fast (12 h), each animal received a subcutaneous injection of compound (15 nmol/rat). The dosing vehicle was 1:1 propylene glycol/20 mmol/L sodium phosphate buffer, pH 7.4. Serial blood samples were collected in EDTA-containing microcentrifuge tubes predose and at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h after subcutaneous administration. Then 0.3 mL of blood was collected at each time point. Blood samples were immediately centrifuged at 4 °C. Plasma was frozen in dry ice and stored at −20 °C. Plasma proteins were precipitated with two volumes of acetonitrile containing an internal standard, mixed by vortexing, and centrifuged at 14 000 rpm for 14 min. Supernatants were transferred to a 96-well plate, and 10 μL was analyzed by LC−MS/MS to determine plasma drug levels. A Waters Acquity UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm) was used. The temperature was maintained at 50 °C, and the flow rate was 0.3 mL/min. The column was equilibrated in 95% buffer A (10 mM

ammonium formate, 0.2% formic acid, water) and 5% buffer B (acetonitrile, 0.2% formic acid). The composition of buffer B was increased to 95% over 2.5 min, held there for 1 min, and returned to 5% buffer B over 0.5 min. The total analysis time was 4.5 min. The UPLC instrument was interfaced to an Applied Biosystems Sciex API-4000 mass spectrometer (Foster City, CA) equipped with a TurboIonSpray ionization source. Ultrahigh purity nitrogen was used as the nebulizing and turbo gas, and regular nitrogen was used as the drying gas. The desolvation gas flow was 648 L/h, and cone gas flow was maintained at 58 L/h. The desolvation temperature was 380 °C, and the source temperature was 120 °C. Observed capillary and cone voltages were 3910 and 45.43 V, respectively. Signals were detected by two multiple reaction monitoring (MRM) transitions. Multiple Intraperitoneal Glucose Tolerance Test in db/db Mice. The ability of 13c and 13l to reduce glucose levels for longer time periods was assessed using a modified multiple IPGTT on T2DM db/db mice.33,48 Briefly, the db/db mice (n = 6/group, 8 weeks, male) were fasted overnight (18 h). At 0.5 h prior to the first intraperitoneal glucose loading (1.0 g/kg), male db/db mice were administered saline (control mice), exendin-4, liraglutide, 13c, or 13l (25 nmol/kg). Blood was collected from tail tip veins at 0, 0.25, 0.5, 0.75, 1, 2, and 3 h after glucose administration, and blood glucose was measured with a blood glucose monitor as described above. After the first IPGTT, glucose loading time points were at 3, 6, 9, 12, 15, 18, and 21 h. The time intervals between glucose loading and blood collection were the same each time. Hypoglycemic Duration Test in db/db Mice. The hypoglycemic efficacies of 13c and 13l were evaluated using a modification of a previously described method using male db/db mice (7 weeks, male).33,49 Under nonfasting conditions with free access to food and water, mice received a single ip injection of saline (control mice), exendin-4 (25 nmol/kg), liraglutide (25 nmol/kg), 13c (25/150 nmol/kg), or 13l (25/150 nmol/kg). A drop of blood was drawn from a tail vein of each animal at different times (0, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 36, and 48 h), and blood glucose levels were measured with a blood glucose monitor. The hypoglycemic durations (time taken for blood glucose to return to levels below 8.35 nmol/L) were also recorded. Preliminary Assessment of Anticoagulant Activity in SD Rats. Male SD rats (200−250 g) were randomly assigned to six groups (n = 6/group). Each animal received vehicle (1:1 propylene glycol/20 mM sodium phosphate, pH 7.4), dicoumarin, 13a, or 13l by intravenous injection (25 or 150 nmol/kg) once daily for 6 consecutive days. On the seventh day, following an overnight fast (12 h), serial blood samples were collected in sodium citrate containing microcentrifuge tubes before dosing and immediately centrifuged at 10000g for 5 min at 20 °C. Plasma was stored at −20 °C until needed for analysis. Anticoagulant activity was measured with a Sysmex CA-7000 coagulometer (Siemens, Germany) according to the manufacturer’s instructions. Chronic in Vivo Studies in db/db Mice. Chronic beneficial effects of 13c were examined in male db/db mice using a previously described method.33 T2DM db/db mice (6 weeks) of both sexes were assigned to three groups (n = 8/group) with matched HbA1c. Two groups received once-daily ip injection of 25 or 50 nmol/kg 13c for 49 days. An amount of 25 nmol/kg liraglutide or saline (control mice) was injected into the other groups. Wild-type nondiabetic C57BL/6 littermates (n = 8/group) were used to normalize the results. HbA1c was measured weekly, and body weight was measured daily. To determine the diabetic status more precisely, mice were subjected to the IPGTT on day 0 and on day 52 (3 days after drug flushing). Briefly, mice in all groups were fasted overnight (18 h) and ip challenged with 1 g/kg glucose. A drop of blood was drawn from a tail vein of each animal 0, 15, 30, 45, 60, 90, and 120 min after glucose administration, and blood glucose levels were measured with a blood glucose monitor. 9966

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Journal of Medicinal Chemistry



Article

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ASSOCIATED CONTENT

S Supporting Information *

Characterization of all dicoumarol GLP-1 conjugates; HPLC chromatograms of tested compounds; concentration responses of dicoumarol GLP-1 conjugates in receptor activation assays; original data for albumin binding; chemical modifications of 9, 10, 11, and 12 investigated in our previous work. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*H.Q.: phone, +86-25-83271302; fax, +86-25-83271297; email, [email protected]. *W.H.: phone, +86-25-83271302; fax, +86-25-83271480; email, [email protected]. Author Contributions †

J.H. and L.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (Grants 81172932 and 81273376), the Natural Science Foundation of Jiangsu Province (Grant BK2012356), and the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (Grant JKGZ201103). The authors thank Dr. Xun Huang at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for his generous help on receptor activation assay.



ABBREVIATIONS USED T2DM, type 2 diabetes mellitus; GLP-1, glucagon-like peptide 1; DPP IV, dipeptidyl peptidase IV; PEG, polyethylene glycol; HSA, human serum albumin; LC−MS/MS, liquid chromatography−tandem mass spectrometry; HPLC, high-performance liquid chromatography; cAMP, cyclic adenosine monophosphate; OGTT, oral glucose tolerance test; AUC, area under the curve; MRT, mean residence time; IPGTT, intraperitoneal glucose tolerance test; TFA, trifluoroacetic acid; FBS, fetal bovine serum; BSA, bovine serum albumin; APTT, activated prothrombin time; PT, prothrombin time; TT, thrombin time



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Journal of Medicinal Chemistry

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