Aug 24, 2009 - Preclinical Candidate Optimization. # Diabetes Drug Discovery. Bristol-Myers Squibb Company Research & Development, Pennington, New Jersey 08534. â Department of Applied Biotechnologies, Bristol-Myers Squibb Company Research & Develo
Aug 29, 2016 - The free fatty acid receptor 4 (FFA4 or GPR120) has appeared as an interesting potential target for the treatment of metabolic disorders. At present, most FFA4 ligands are carboxylic acids that are assumed to mimic the endogenous long-
Aug 29, 2016 - ... cruzi, Leishmania amazonensis and Herpes Simplex Virus .... Nystrom , Vincent Rash , Thomas Rimele , Shane Roller , Sean Ross.
Chem. Lett. , 2014, 5 (4), pp 368â372. DOI: 10.1021/ml400491k. Publication Date (Web): January 27, 2014 ... Design and synthesis of novel Î´ opioid receptor agonists with an azatricyclodecane skeleton for improving blood-brain barrier penetration.
The results showed that the TPA molecule and the teleocidin sofa form could interact with the com- mon receptor through three hydrogen bonds, and the sofa.
Jun 1, 2011 - Dilip K. Tosh, Khai Phan, Francesca Deflorian, Qiang Wei, Zhan-Guo Gao, and Kenneth A. Jacobson*. Molecular Recognition Section ...
Jun 7, 2018 - Novel peptidic dual agonists of the glucagon-like peptide 1 (GLP-1) and glucagon receptor are reported to have enhanced efficacy over pure ...
Jun 7, 2018 - Currently there are more than 400 million patients living worldwide with .... glucagon and GIP(1â42) bear hydrophobic residues in this position (Met27 ..... peptide, 0, 1, 2, 4, 8, 22, AUC0â22 (hÂ·ng/mL) ...... Soni, H. Peptide-Base
Nov 4, 2016 - We have examined analogues of glucagon-like peptide-1 (GLP-1) containing Î²-amino acid residues in place of native Î± residues at selected sites and found that some analogues differ from GLP-1 in terms of their relative abilities to pro
Nov 4, 2016 - We have examined analogues of glucagon-like peptide-1 (GLP-1) containing Î²-amino acid residues in place of native Î± residues at selected sites and found that some analogues differ from GLP-1 in terms of their relative abilities to pro
7788 J. Med. Chem. 2009, 52, 7788–7799 DOI: 10.1021/jm900752a
Eleven Amino Acid Glucagon-like Peptide-1 Receptor Agonists with Antidiabetic Activity Claudio Mapelli,† Sesha I. Natarajan,† Jean-Philippe Meyer,† Margarita M. Bastos,† Michael S. Bernatowicz,† Ving G. Lee,† Jelka Pluscec,† Douglas J. Riexinger,† Ellen S. Sieber-McMaster,† Keith L. Constantine,† Constance A. Smith-Monroy,# Rajasree Golla,# Zhengping Ma,# Daniel A. Longhi,# Dan Shi,§ Li Xin,# Joseph R. Taylor,# Barry Koplowitz,z Cecilia L. Chi,z Ashish Khanna,z Gordon W. Robinson,# Ramakrishna Seethala,# Ildiko A. Antal-Zimanyi,# Robert H. Stoffel,§ Songping Han,# Jean M. Whaley,# Christine S. Huang,z John Krupinski,*,# and William R. Ewing*,‡ Department of Applied Biotechnologies and ‡Discovery Chemistry and zPreclinical Candidate Optimization and #Diabetes Drug Discovery, Bristol-Myers Squibb Company Research & Development, Pennington, New Jersey 08534, and †Department of Applied Biotechnologies, Bristol-Myers Squibb Company Research & Development, Princeton, New Jersey 08543
Received May 29, 2009
Glucagon-like peptide 1 (GLP-1) is a 30 or 31 amino acid peptide hormone that contributes to the physiological regulation of glucose homeostasis and food intake. Herein, we report the discovery of a novel class of 11 amino acid GLP-1 receptor agonists. These peptides consist of a structurally optimized 9mer, which is closely related to the N-terminal 9 amino acids of GLP-1, linked to a substituted C-terminal biphenylalanine (BIP) dipeptide. SAR studies resulted in 11-mer GLP-1R agonists with similar in vitro potency to the native 30-mer. Peptides 21 and 22 acutely reduced plasma glucose excursions and increased plasma insulin concentrations in a mouse model of diabetes. These peptides also showed sustained exposures over several hours in mouse and dog models. The described 11-mer GLP-1 receptor agonists represent a new tool in further understanding GLP-1 receptor pharmacology that may lead to novel antidiabetic agents.
Introduction Type 2 diabetes has been increasing worldwide at an alarming rate. It has been projected that the number of people suffering from diabetes will rise to 366 million by 2030, more than doubling the prevalence of 171 million patients estimated for the year 2000.1 The prevalence and severity of diabetes has resulted in an increased need for new therapeutic agents that have the potential to counter the progressive hyperglycemia, weight gain, and loss of pancreatic β cell function that typify the natural history of the disease and ultimately result in significant morbidity and mortality. Glucagon-like peptide 1 (GLP-1a) is a gastrointestinal hormone that exists predominantly as a 30 amino acid, C-terminally amidated peptide (GLP-1(7-36)NH2) or as the equally active, glycine-extended form, GLP-1(7-37), either of which is a functional agonist of the GLP-1 receptor, a class II GPCR. The importance of GLP-1 and its receptor in regulating glucose homeostasis and food intake supports the view that GLP-1 receptor agonists have the potential to improve on the current standard of care in treating type 2 diabetes. Release of GLP-1 *To whom correspondence should be addressed. For J.K.: phone, 609-818-4977; fax, 609-818-3239; e-mail, [email protected] For W.R.E.: phone, 609-818-3483; fax, 609-818-3331; e-mail, william. [email protected] a Abbreviations: GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; BIP, biphenylalanine; DPP4, dipeptidyl peptidase IV; GPCR, G-protein-coupled receptor; cAMP, cyclic adenosine monophosphate or adenosine 30 ,50 -cyclic monophosphate; rmsd, root-mean-square deviation; IPGTT, intraperitoneal glucose tolerance test; ANOVA, analysis of variance; Cmax, maximum plasma concentration; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LLQ, lower limit of quantitation.
Published on Web 08/24/2009
from intestinal L-cells in response to oral food intake potentiates insulin secretion from pancreatic β-cells as part of the “incretin effect”.2-4 However, unlike direct administration of insulin, GLP-1 receptor agonism has a low potential to induce hypoglycemia because both GLP-1-dependent stimulation of insulin release and inhibition of glucagon secretion from the pancreas are glucose-dependent and do not occur when plasma glucose decreases below normal concentrations.5 Importantly, GLP-1 can also inhibit both gastric emptying6 and food intake,7 resulting in weight loss following chronic administration in animal models8 and obese, type 2 diabetic patients.9 In animal models, GLP-1 has also been shown to preserve or increase β-cell mass through inhibition of apoptosis, stimulation of proliferation, and possibly, stimulation of β-cell differentiation.10 Continuous coverage with a GLP-1 receptor agonist is the optimal pharmacokinetic profile, as type 2 diabetic patients infused with GLP-1 for 24 h showed more beneficial effects on fasting plasma glucose when compared to patients infused for 16 h.11 Endogenous GLP-1, however, is short-lived in vivo, with a half-life of ∼2 min.12 Therefore, in a broad patient population requiring chronic treatment, the unmodified GLP-1 peptide is not suitable as a therapeutic agent. The major degradation pathway of GLP-1 is cleavage of the HisAla dipeptide from the N-terminus abolishing agonist activity, a process mediated by dipeptidyl peptidase IV (DPP4).13 However, DPP4 has other natural substrates in addition to GLP-1, and the plasma concentrations of GLP-1 achieved by DPP4 inhibition are limited, resulting in a somewhat distinct clinical profile. Importantly, DPP4 inhibitors are weight neutral, while GLP-1 receptor agonists promote weight loss.14 r 2009 American Chemical Society
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Scheme 1. Solid Phase Suzuki Synthesis of a Biphenylalanine Residue in Positions 10 and 11a
a (a) 20% piperidine/DMF; (b) Boc-4-I-Phe-OH, BOP, DIEA in DMF; (c) Fmoc-amino acid/DIC/HOAt, NMP; (d) Pd(0) cat., 25% DIEA/NMP, 85-90 °C, arylboronic acid; (e) TMS-OTf/2,6-lutidine/CH2Cl2 (1:1:3); (f) 20% MeOH/DMF.
All of the GLP-1 receptor agonists approved or reported to be in clinical development are based on the amino acid sequence of GLP-1 or the 39-amino acid sequence of exendin-4, a GLP-1R agonist isolated from the saliva of the gila monster, Heloderma suspectum.15 Synthetic exendin-4 (exenatide, Byetta) is a very potent, DPP4-resistant GLP-1 receptor agonist and is the first agent in this class approved for the treatment of type 2 diabetes. However, the subcutaneous formulation of exenatide is administered twice daily16 and considerable effort has been devoted to enhancing the pharmacokinetic properties of exenatide and other peptidic or protein-based GLP-1R agonists in development.17 Although a significant discovery, the nonpeptidic GLP-1R agonists disclosed to date are not sufficiently potent to be suitable for clinical development.18,19
Scheme 2. Preparation of Fmoc-(S)-(20 -methyl)biphenylalanine and Fmoc-(S)-(20 -ethyl-40 -methoxy)biphenylalaninea
a (a) K2CO3, Pd(PPh3)4, toluene, 80 °C, 2-Me-phenylboronic acid or 2-Et-phenylboronic acid; (b) THF/MeOH (3:1), NaOH; (c) HCl/ CH2Cl2; (d) THF/H2O (4:1), Fmoc-OSu.
Scheme 3. Preparation of 2-Ethyl-4-methoxyphenylboronic Acid, Routes A and Ba
Chemistry Amino Acid Synthesis. The described biphenylalanines (2amino-3-(biphenyl-4-yl)propanoic acids, BIP) were synthesized on a solid support using Rink resin or by using solution phase chemistry. In Scheme 1, the process to synthesize position 10 or 11 biphenylalanines is described. 4-Iodophenylalanine is coupled to Rink resin and is subjected to Suzuki coupling conditions using either tetrakis(triphenylphospine)palladium(0) or palladium(II) acetate/2-(dicyclohexylphosphino)biphenyl catalyst. In this process, a 10-fold excess of boronic acid and heating to 80 °C is needed for efficient coupling. In a similar fashion (Scheme 1), position 10 biphenylalanines could be synthesized via coupling iodophenylalanine to the desired position 11-amino acid on the Rink resin followed again by Suzuki coupling conditions. Once identified, optimal position 10 and position 11 biphenylalanines were synthesized individually using solution phase chemistry following the procedures shown in Scheme 2. Both procedures start with the conversion of the phenol of Boc-L-Tyr methyl ester to the corresponding triflate. In the synthesis of the optimal position 11 amino acid, the resulting product was coupled under Suzuki conditions with 2-methylphenylboronic acid. The ester of the resulting product was hydrolyzed, and then the Boc group
was converted to the Fmoc group in a two step sequence to yield (S)-2-(Fmoc)-3-(20 -methylbiphenyl-4-yl)propanoic acid (BIP-(20 -Me)). Also shown in Scheme 2, an analogous procedure was used to synthesize (S)-2-(Fmoc)-3-(20 -ethyl-40 -methoxybiphenyl-4-yl)propanoic acid (BIP-(20 -Et-40 -OMe)), which was found to be the optimal amino acid at position 10. The synthesis of 2-ethyl-4-methoxyphenylboronic acid needed for the preparation of BIP-(20 -Et-40 -OMe) is shown in Scheme 3. The desired boronic acid could be conveniently prepared from either 2-bromo-5-methoxybenzaldehyde or 3ethylphenol. Fmoc-(S)-2-Fluoro-R-methylphenylalanine, used in position 6 of the 11-mers, was prepared using an oxazolone alkylation route, followed by enzymatic resolution of the resulting racemic amino acid using carboxypeptidase A (Scheme 4). This amino acid is also commercially available in chiral form. Fmoc-(S)-2,6-difluoro-R-methylphenylalanine was prepared in a similar manner.
7790 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Scheme 4. Preparation of Fmoc-(S)-2-fluoro-R-methylphenylalaninea
Figure 1. Amino acid sequence of GLP-1(7-36) amide.
Peptide Synthesis. Peptides were prepared by solidphase synthesis using a NR-Fmoc/tert-butyl protection strategy on 4-(20 ,40 -dimethoxyphenyl-Fmoc-aminomethyl)phenoxy (Rink) resin.20 After assembling on solid phase, the resulting protected 11-mers were cleaved from the resin, deprotected using either TFA/TIS/water or the TFA-based reagent K.21 Coupling to R-methylphenylalanine and fluorinated analogues in position 6 required use of a large excess (10-fold) of Fmoc-Thr(OtBu)-OH and HOAt/DIC in NMP for the reaction to go to completion. Methods for purifications by preparative RP-HPLC, biology methods, and characterization data are in the Experimental Section and Supporting Information. Results and Discussion Since the discovery of GLP-1 (Figure 1), significant advances have been made in the understanding of the structural requirements for the functional activity of this endogenous peptide. As well, modifications to improve the biological halflife of GLP-1 have been discovered through key amino acid substitutions, modifications, and conformational constraints linking amino acid side chains. SAR studies focused on the determination of the key residues important for the binding and function of GLP-1 demonstrated that the minimal sequence that retained functional agonist activity, i.e., GLP-1R-mediated stimulation of cAMP accumulation, is the 7-34 sequence of GLP1(7-37).22,23 From alanine scans, the amino acids of greatest importance were determined to be those at positions 7, 10, 12, 13, 15, 19, 21, 28, and 29.24,25 Residues His-7 and Asp-15, in particular, appear to be necessary and critical for functional activity.26 With most of the sequence and with many amino acids needed for agonist activity, approaches to improve on the GLP-1 structure have involved specific amino acid substitutions and modifications to less critical amino acid residues of the 30 amino acid sequence. Stabilization of GLP-1 to the primary cleavage event by DPP4 was accomplished through the recognition that Ala-8 could be replaced with other amino acids including Aib, Gly, Ser, and Thr without significant loss
Mapelli et al.
in functional activity.27,28 Among these, the GLP-1 analogue with Aib was found to be the most stable to DPP4 cleavage and to have potency similar to native GLP-1. Other modifications were found that increased biological half-life, with one of the most significant identified by the Novo Nordisk group, showing that γ-glutamyl-mediated acylation with a fatty acid of either a native or installed Lys residue across the GLP-1 sequence gave 31-mers with significant improvements in halflife.29,30 Their research ultimately resulted in the discovery of liraglutide which has completed phase III trials. Through all of the structural advances, a model had developed in which three regions of the GLP-1 peptide were determined to be necessary for agonist activity at the GLP-1 receptor.31 Herein we report the discovery of modified GLP-1 peptides that represent a new minimal sequence for highly potent full agonists of the GLP-1 receptor, a class II GPCR. Our approach started with the iterative addition of one amino acid to the N-terminal 9-mer sequence of GLP-1. This 9-mer sequence was chosen on the basis of research showing that five of the amino acids critical for binding or functional activity were located in this region and were thought to represent one of the domains important for interactions with the GLP-1R. Starting with this sequence, mono- and dipeptide sequences were added and assessed in vitro for functional activity by measuring cAMP accumulation in Chinese hamster ovary cells overexpressing the human GLP-1R. Through our initial scan, it was found that an 11-mer peptide containing two biphenylalanine residues (BIP) at the C-terminus, 1, is a submicromolar (545 ( 117 nM) full agonist for the GLP-1 receptor in the cAMP accumulation assay (Table 1), and the functional activity can be inhibited by the known GLP-1R antagonist exendin-4(9-39) (Figure 2).32 The concentration of exendin-4(9-39) that causes a 2-fold increase in the EC50 of peptide 1 was estimated to be 37 nM (from the curve fitting, pA2 = -1.57 ( 0.16 nM), and the Schild slope was not statistically significantly different from 1, consistent with simple competitive inhibition by exendin4(9-39) in the functional assay. Peptide 1 also displaced 125 I-GLP-1 in a competitive radioligand binding assay (data not shown), consistent with literature reports that five of the first nine amino acids of GLP-1 are important for receptor binding.24,25 In the following SAR studies, primary emphasis was placed on optimizing potency in the functional assay because the 11-mers are structurally distinct GLP-1R agonists. Both BIP residues were determined to be critical for activity, as the 10-mer 2 was completely inactive (Table 1). As well, the C-terminal carboxamide was important for potency, as the corresponding acid (peptide 3) was found to be inactive. After the identification of 1, peptides were synthesized in which BIP was kept constant at either position 10 or 11 (equivalent to position 16 or 17 in GLP-1(7-36) amide) and the corresponding open position was scanned with selected amino acids (Figure 3). From this study, no dipeptide was identified that was as potent as 1. From the collection, only one BIP replacement at position 10 (2-naphthyl-Ala) was found that resulted in weak agonist activity (9000 nM). However, replacements for the BIP in position 11 did identify other modestly active sequences (BIP with 2-naphthyl-Ala, 4MePhe, 4-nitroPhe or Tyr) with agonist activity of approx 10 000 nM. With peptide 1 remaining as the most active 11-mer, attempts to increase potency were envisioned through substitutions on the phenyl rings of the BIPs (Table 1). A methyl
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Table 1. 11-mer GLP-1R Agonists: Positions 10 and 11 SAR
Figure 2. Concentration-response of peptide 1-mediated stimulation of cAMP accumulation was measured in CHO cells overexpressing the human GLP-1 receptor in the presence of the concentrations of the antagonist, exendin-4(9-39), indicated in the legend. All of the data were fit simultaneously to a modified Gaddum-Schild model (GraphPad Prism software, version 4.03) to estimate the concentration of the antagonist that caused a 2-fold increase in the EC50 of peptide 1.
walk around the distal ring of the position 11 BIP (peptides 5, 6, 8) resulted in analogues that were essentially equipotent to 1. Likewise, the addition of an electron withdrawing fluoro (peptide 7) or an electron donating methoxy (peptide 9) did not affect activity. While no improvements in functional activity were achieved through these modifications, many groups were identified that retained activity. In contrast, substitutions to the distal phenyl ring of the BIP in position 10 had a larger effect on activity. Notably, addition of a methyl to the 20 -position increased activity and could be paired with any of the position 11 substituted BIPs to improve functional activity to the 100 nM range (peptides 10, 11, 12). Other 20 substitutions were explored. Peptide 13, with an ethyl in the 20 position, increased activity to 27 nM, a 20fold improvement over 1. Likewise, the analogue with the
Figure 3. 11-mer SAR: scan of amino acids with BIP in position 10 or 11. Analogues with weak GLP-1R agonist activity are shown with EC50 values in parentheses.
20 -ethyl-BIP in 10 and the 20 -methyl-BIP in 11 was found to be equally potent (22 nM, peptide 14). Peptide 15, with 20 -propylBIP in position 10, is less active with EC50 = 93 nM. Other substitutions in the distal ring of the position 10 BIP that increased functional activity were also found. Peptide 16 with the 20 -methyl-40 -methoxy substitution showed an increase in functional activity relative to peptide 5, whereas the 30 -methoxy, 17 (1000 nM), is somewhat less active relative to peptide 5 in comparison. From the SAR studies on the position 10 BIP, an analogue combining the optimized substitutions was synthesized. The resulting analogue containing 20 -ethyl-40 methoxy-BIP in position 10, peptide 18, is a 7 nM full agonist for the GLP-1 receptor. With the optimization of the BIPs in positions 10 and 11 yielding analogues with nearly 100-fold improvements in GLP-1 receptor functional activity over the lead peptide 1,
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further gains in potency were attempted through modifications of the amino acids in positions 1-9. Studies on the conformation of GLP-1 have identified several regions that have R-helical character. These are found between residues Thr13-Glu20 and Ala24-Gly35, with the latter being more stable as determined by solution NMR studies. However, by contrast the N-terminal His7-Thr 13 was found to be a random coil structure in solution. The described 11-mers can then be characterized as overlaying with both the domains containing the random coil and R helix.33 Recently, studies on the receptor bound conformation of GLP-1 have shown that the N-terminal R helix extends over residues 11-2134 (which would start at position 5 in the described 11-mers). In the design of truncated GLP-1 analogues, it was recognized that conformational order would be needed to provide 11-mers with GLP-1 like potency. In an attempt to further induce helical structure in the above-described 11-mer peptides, R-methylamino acids were tried in several positions. Peptide 19 (Table 2), with an R-methyl-Phe in position 6, showed modest gains in potency relative to peptide 18. When this finding was paired with the structurally enforcing and DPP4 stabilizing Aib in position 2, a subnanomolar peptide, 20 (EC50=0.28 nM), was obtained. The 11-mer, peptide 20 is within 10-fold of the activity of the 30 amino acid native peptide, GLP-1. Further improvements in potency were realized through SAR efforts focusing on substituting the phenyl of R-methyl Phe. Of the substitutions explored, fluoro groups at the 2 position or the 2 and 6 positions were found to substantially improve activity. Peptide 21, containing an Table 2. 11-mer GLP-1R Agonists: Positions 2 and 6 SAR His-Xaa2-Glu-Gly-Thr-Xaa6-Thr-Ser-Asp- BIP(20 -Et, 40 -OMe)BIP-(20 -Me)-NH2 hGLP-1R cAMP peptide no. Xaa2 Xaa6 EC50 (nM) 19 20 21 22
R-methyl-2-fluoro-Phe, and peptide 22, containing R-methyl2,6-difluoro-Phe, show significant improvements in functional activity and were found to have similar potency to GLP-1. Using 2D 1H-1H NMR methods, the solution conformation of peptide 21 was determined and the NMR structure ensemble is shown in Figure 4. This ensemble reflects a population of ordered conformations for peptide 21, with residues 6-11 forming a 310 helix showing some distortion from the canonical conformation for residues 9-11. There is a kink at position 2, and residues 3 and 4 could be characterized as a distorted type I turn or a type VIIa turn. The region around the R-methyl-2-fluoro-Phe in position 6 shows the formation of an incipient 310 helix, most likely in synergy with the Aib in position 2. Analogues lacking R-methylation at position 6 exhibited signatures of nascent turns or helices in their NMR spectra but did not yield sufficient numbers of NOE distance restraints to produce well-defined conformations. Peptide 21 was found to be highly selective (EC50 or IC50 values of >10 μM) against several related class II GPCRs: glucagon, glucose-dependent insulinotropic (GIP), the pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and parathyroid hormone (PTH) receptors. In addition, peptide 21 was also profiled against 31 different receptors, enzymes, channels, and transporters (CEREP screen) and was determined to be >10000fold selective against all of these targets. In preparation for in vivo testing, peptides 21 and 22 were profiled in cells expressing the mouse GLP-1 receptor. The potency of native GLP-1 was not significantly different at the two receptors. However, peptide 21 showed a ∼30-fold decrease in functional potency with an EC50 value of 2.4 ( 0.8 nM (mean ( SEM; n=11) versus 0.087 ( 0.01 nM (n=15) in the mouse versus human GLP-1R cAMP accumulation assays, respectively. For peptide 22, the EC50 values for activation of the mouse versus human GLP-1R were 0.8 ( 0.17 nM (n=9) and 0.12 ( 0.02 nM (n=12), respectively. The potencies of both peptides 21 and 22 were statistically significantly different (p < 0.05) when compared at receptors across species but not when compared to each other at a single
Figure 4. Stereoview of an ensemble of 20 NMR structures of peptide 21 computed using 62 sequential, 67 medium-range, and 8 long-range NOE restraints. The average backbone atom rmsd to the mean structure is 0.29 A˚, and the average all-heavy-atom rmsd to the mean structure is 1.00 A˚. Atoms are color-coded according to type; hydrogens are not shown. His1 is near the bottom on the right side of the image.
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Figure 5. Effects of a single subcutaneous dose of peptides 21 and 22 in an IPGTT in male ob/ob mice. Peptide (dose in nmol/kg indicated in parentheses in the legends) or vehicle (1:1 propylene glycol/20 mM sodium phosphate, pH 7.4) was administered to groups of six fasted male ob/ ob mice 30 min prior to the intraperitoneal administration of 2 g/kg glucose at time 0. The time course for plasma glucose changes during the IPGTT from two separate studies are shown in panels A and C, and the corresponding insulin time courses are shown in panels B and D.
receptor (p > 0.05). The species dependence of the activity of the 11-mers may reflect binding of the unnatural amino acids in positions 10 and 11 to a region of the receptor which can vary across species because it is not essential for binding of the native ligand. Peptide 21 was profiled in vivo in the insulin resistant ob/ ob mouse following a single subcutaneous dose of 30 or 300 nmol/kg with 1 nmol/kg exendin-4 as a positive control (Figure 5A,B). The acute efficacy of the peptides was assessed using a glucose tolerance test (GTT) initiated by intraperitoneal (ip) injection of 2 g/kg glucose 30 min after subcutaneous administration of the peptides or vehicle. Figure 5A shows the time course for plasma glucose changes during the IPGTT. Plasma glucose values in all three peptide-treated groups were statistically significantly lower than those in the vehicle group at each time point from 30 to 180 min after the glucose challenge (ANOVA with Dunnett’s test, p < 0.001 vs vehicle at t = 30-180 min). The plasma glucose values were not statistically significantly different at any time point following administration of either 300 nmol/kg peptide 21 or 1 nmol/kg exendin-4 (Figure 5A). The decreases in plasma glucose were accompanied by increases in plasma insulin concentrations, consistent with a GLP-1-dependent mechanism (Figure 5B). The time courses for plasma insulin concentrations were again very similar following treatment with 300 nmol/kg peptide 21 or 1 nmol/kg exendin-4. With either of these treatments, the plasma insulin concentrations were statistically significantly greater than those of the vehicle group at both the 30 min (p < 0.01) and 60 min (p < 0.05) time points based on an ANOVA followed by Dunnett’s post hoc test. Peptide 21 exhibits a similar efficacy profile but is considerably less potent than exendin-4 in the ob/ob mouse IPGTT model. Peptides 21 and 22 also produced similar glucose lowering to each other and stimulated insulin secretion when compared in the IPGTT model at a dose of 300 nmol/kg (Figure 5C and Figure 5D). Consistent with their similar in vitro potencies, it was not possible to differentiate peptides 21 and 22 in the ob/ob IPGTT model.
Figure 6. Pharmacokinetics of peptides 21 and 22 in the ob/ob mouse. The vehicle was 1:1 propylene glycol/20 mM sodium phosphate, pH 7.4. Samples were collected at 1, 2, 4, 6, 8, and 24 h postdosing and were analyzed by LC-MS/MS.
The pharmacokinetics of peptides 21 and 22 were measured in the ob/ob mouse, the pharmacodynamic model (Figure 6). The peptides were administered via subcutaneous injection of 300 nmol/kg in 1:1 propylene glycol/20 mM sodium phosphate buffer, pH 7.4. The Cmax values were greater than 2000 nM for peptides 21 and 22, and both peptides achieved similar measurable exposures over 24 h in ob/ob mice. Plasma concentrations of exendin-4 were below the lower limit of quantitation (∼5 nM) of the LC-MS method following administration of the 1 nmol/kg dose used in the efficacy study, underscoring the marked difference in potency between these peptides (Figure 5). Even accounting for decreased potency at the mouse GLP-1 receptor, these relatively high efficacious plasma concentrations suggest that improvements in the in vivo potency will be required to produce an 11-mer that could be administered as a ∼1 mg dose in human diabetic patients, similar to liraglutide.35 The pharmacokinetic properties of peptides 21 and 22 were also investigated in a normal dog model. Subcutaneous administration of 67 μg/kg (∼45 nmol/kg) in 1:1 propylene glycol/20 mM sodium phosphate buffer, pH 7.4, resulted in measurable exposure over a 24 h period for peptide 22, but peptide 21 could not be detected beyond the 8 h time point (Figure 7). This was the first significant difference in the
7794 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Figure 7. Pharmacokinetics of peptide 21 and peptide 22 in the dog. The figure shows the plasma concentration-time profiles following subcutaneous (sc) administration in male beagle dogs (67 μg/kg, n = 3). The dosing vehicle was 1:1 propylene glycol/20 mM sodium phosphate buffer, pH 7.4. Samples were collected at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 24, and 30 h postdosing and were analyzed by LC-MS/ MS. The plasma concentration of peptide 21 was below the LLQ at 24 and 30 h.
profiles of these two peptides. Peptide 22 had a mean residence time of 20 ( 5 h, suggesting that this peptide may potentially meet an important PK criterion for a once a day agent. Moreover, the peptide shows a relatively low peak to trough ratio. This could be significant if this PK profile were to translate to humans because rapid increases in plasma concentration and a high Cmax appear to contribute to the doselimiting side effects of nausea and vomiting reported for peptide GLP-1 receptor agonists in the clinic.36-38 In conclusion, we have discovered a novel class of small peptide agonists for the GLP-1 receptor. Through replacement of the C-terminal 21 amino acids with a biphenylalanine dipeptide followed by sequential optimization of several key N-terminal amino acid residues, 11-mer peptides were discovered that have in vitro functional activity similar to that of GLP-1 while maintaining selectivity versus other related class II GPCRs. Peptides 21 and 22 were shown to stimulate insulin secretion and reduce glucose excursions in vivo in a mouse model of diabetes, consistent with the expected mechanism of action for a GLP-1R agonist. In addition, these 11-mers show an enhanced pharmacokinetic half-life relative to GLP-1. In particular, peptide 22 displays favorable pharmacokinetic properties in mouse and dog PK models, demonstrating a low peak to trough with 24 h of exposure. The 11-mers represent a unique tool for further investigating the pharmacology of the GLP-1 receptor and, with additional improvements of in vivo potency, could represent a new series of clinical agents for the treatment of type 2 diabetes. Experimental Section Materials. NR-Protected amino acids and the 4-(20 ,40 -dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin) were purchased from Novabiochem (San Diego, CA). The following reagents were purchased from the commercial sources indicated: trifluoroacetic acid (TFA, EMD Chemicals); N,N-diisopropylcarbodiimide (DIC), N,N-diisopropylethyl amine (DIEA), Nbromosuccinimide (NBS), piperidine, acetic anhydride, and triisopropylsilane (TIS) (Aldrich, Milwaukee, WI); dichloromethane (CH2Cl2, Mallinckrodt Baker, Inc.); N-methylpyrrolidone (NMP) and N,N-dimethylformamide (DMF) (Burdick and Jackson); 1-hydroxy-7-azabenzotriazole (HOAt), 2-(1Hbenzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetra-
Mapelli et al.
methyluronim hexafluorophosphate (HATU) (Applied Biosytems, Foster City, CA); HPLC-grade acetonitrile (Mallinckrodt Baker, Inc.). Solution Synthesis of Substituted Biphenylalanines (2-Amino3-(biphenyl-4-yl)propanoic Acid or BIPs). The biphenylalanine derivatives described in this work were synthesized using the procedures exemplified below, starting from the commercially available Boc-tyrosine methyl ester to prepare the triflate and using the appropriate boronic acid to produce the desired biphenylalanine analogues. When a required boronic acid was not available from commercial sources, its synthesis was performed as exemplified below. Synthesis of (S)-2-(9H-fluoren-9-yl)methoxy)carbonylamino)3-(20 -methylbiphenyl-4-yl)propanoic Acid or Fmoc-(20 -Me)-BIPOH. a. Preparation of Boc-L-tyrosine O-Triflate Methyl Ester. To a solution of Boc-tyrosine methyl ester (126 mmol, 37 g) and pyridine (25.4 mL, 314 mmol) in CH2Cl2 (100 mL), at -15 °C under N2, was added slowly triflic anhydride (25.4 mL, 151 mmol). The solution was stirred at -15 °C for 15 min, and after this time HPLC analysis indicated that the reaction was complete. To the reaction was added water (150 mL). The cooling bath was removed, and the layers were separated. The organic layer washed with 0.5 M aqueous NaOH (2 150 mL) and 15% aqueous citric acid (2 150 mL). The organic layer was dried over MgSO4, filtered, and concentrated to give the crude product as a red oil. The crude product was used “as is” in the next step. b. Preparation of (S)-2-(tert-Butoxycarbonylamino)-3-(20 methylbiphenyl-4-yl)propanoic Acid Methyl Ester. A degassed suspension of o-tolylboronic acid (140 mmol, 19.0 g), K2CO3 (175 mmol, 24.1 g), and tetrakis(triphenylphosphine)palladium (0) (4.0 mol, 4.6 g, 0.034 equiv) in toluene (580 mL) is heated to 80 °C. To the resulting suspension was added the crude Boc-Ltyrosine O-triflate methyl ester from the previous reaction step (above) dissolved in toluene (70 mL). The resulting reaction mixture was maintained at 80 °C under N2 for 3 h. After this time, the reaction mixture was cooled to room temperature and then filtered through a plug of Celite. The filtrate was washed with 0.5% aqueous NaOH (2 150 mL) and 15% aqueous citric acid (2 150 mL). The organic layer was then dried over MgSO4, filtered, and concentrated. The obtained crude mixture was purified by silica gel column chromatography, eluting with EtOAc/heptane (1:9) [the crude mixture was preabsorbed on silica gel (2 g silica gel/g crude mixture), 1:35 mixture/silica gel used for the column], and yields varied from 50% to 80%. c. Preparation of (S)-2-(tert-Butoxycarbonylamino)-3-(20 -methylbiphenyl-4-yl)propanoic Acid. (S)-2-(tert-Butoxycarbonylamino)3-(20 -methylbiphenyl-4-yl)propanoic acid methyl ester (120 mmol, 44.5 g) is dissolved in a solution of MeOH (147 mL) and THF (442 mL). To the resulting solution at room temperature was added 1 N NaOH (147 mmol, 147.4 mL). HPLC analysis indicated that the reaction was complete after 1 h. After this time, the reaction mixture was concentrated at room temperature to remove the MeOH and THF. The resulting solution was partitioned between water (500 mL) and Et2O (300 mL). The layers were separated, and the aqueous layer was acidified with 1 N HCl (160 mL) and then extracted with Et2O (2 250 mL). The combined Et2O extracts were dried over MgSO4, filtrated, and concentrated, resulting in 41.5 g of title compound. d. Preparation of (S)-2-((9H-Fluoren-9-yloxy)carbonylamino)3-(20 -methylbiphenyl-4-yl)propanoic Acid. To a solution of (S)2-(tert-butoxycarbonylamino)-3-(20 -methylbiphenyl-4-yl)propanoic acid (117 mmol, 41.5 g) in CH2Cl2 (1 L) kept at room temperature was slowly bubbled in gaseous HCl. After 5 min, a white solid started to precipitate. After 2 h, the reaction was shown to be complete by HPLC. The reaction mixture was then concentrated, and the resulting residue was redissolved in THF (600 mL). To the resulting solution was added water (150 mL) followed by the slow portionwise addition of solid NaHCO3
until the pH of the mixture was basic. A white solid precipitated out, and Fmoc-OSu (115 mmol, 38.9 g) was then added. The resulting reaction mixture was stirred at room temperature, and after 1 h, a homogeneous biphasic solution was obtained. Stirring was continued at room temperature for an additional 16 h. After this time, the layers were separated. The THF layer was acidified with 2 N aqueous HCl (58 mL) and then diluted with EtOAc (400 mL). The layers were separated, and the organic layer was washed with water (2 100 mL), dried over MgSO4, filtered, and concentrated. The crude product was purified using silica gel column chromatography using CH2Cl2 as eluant until most of the fast running impurities had been removed. The eluant was then changed to 25% EtOAc in heptane containing 1% acetic acid. The yield was >90% for the three steps. (S)-2-((9H-Fluoren-9-yloxy)carbonylamino)-3-(20 -ethyl-40 -methoxybiphenyl-4-yl)propanoic Acid or Fmoc-BIP-(20 -Et-40 -OMe). Method A. Synthesis of (2-Ethyl-4-methoxy)phenylboronic Acid. a. Preparation of (2-Bromo-5-methoxy)styrene. A suspension of methyltriphenylphosphonium bromide (199.5 g, 0.465 mol) in dry THF (800 mL) was purged for 10 min and cooled to 10 °C. Then 2.75 M n-BuLi in hexanes (169 mL, 0.465 mol) was added slowly over 30 min. The resulting solution was stirred for 1 h. To the reaction mixture was slowly added over a period of 30 min a solution of (2-bromo-5-methoxy)benzaldehyde (100 g, 0.465 mol) in dry THF (300 mL). After the addition was complete, the reaction mixture was stirred for 1 h. Petroleum ether (2 L) was added, and the reaction mixture was stirred for an additional 30 min and then filtered through a silica gel pad. The pad was washed with Et2O. The combined organic washes were concentrated with the temperature kept below 30 °C. The resulting the crude product was purified by silica gel column chromatography, using 100% petroleum ether as eluent. Yield: 92 g, 90%, as pale-yellow liquid. b. Preparation of (4-Bromo-3-ethyl)anisole. 2,20 -Bipyridyl (24.3 g, 0.15 mol) and (2-bromo-5-methoxy)styrene (0.31 mol, 65 g) were dissolved in EtOAc (650 mL), and the resulting solution was cooled to 0 °C. The solution was purged with nitrogen, and 10% palladium on carbon (16.25 g, 25%) was added under a stream of nitrogen. The reaction mixture was stirred under 2 kg pressure in a Parr shaker for 3 days under hydrogen. The reaction progress was monitored by HPLC. Upon completion, the reaction mixture was filtered through Celite, and the filtrate was washed with 5% aqueous KHSO4, dried over Na2SO4, filtered, and concentrated, keeping the temperature below 30 °C. Yield: 60 g, 91%, as pale-yellow liquid. LC-MS: (M þ H)þ at m/z 214, 216. 1H NMR (300 MHz, CDCl3): δ 7.37 (dd, 1H, J=3, 10 Hz); 6.77 (d, 1H, J=3 Hz); 6.65 (dd, 1H, J=3, 10 Hz); 3.80 (s, 3H); 2.77 (2H, q, J=7.6 Hz); 1.24 (3H, t, J=7.6 Hz). c. Preparation of (2-Ethyl-4-methoxy)phenylboronic Acid. A solution of (4-bromo-3-ethyl)anisole (94 g, 0.437 mol) in THF (900 mL) was cooled to -78 °C, and n-BuLi (249 mL, 0.55 mol) was added dropwise while maintaining the same temperature. Stirring was continued for 1 h at -78 °C, and tri-n-butyl borate (177 mL, 0.655 mol) was then added slowly at -78 °C. The cooling bath was removed, and the reaction mixture was allowed to warm to 0 °C and was then quenched with 1.5 N hydrochloric acid at 0 °C. The organic layer was separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated. The resulting residue was stirred in petroleum ether for 30 min, and the solid obtained was collected by filtration and then dried under vacuum. Yield: 65 g, 82%, as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 7.80 (br s, 2H); 7.41 (d, 1H, J=9); 6.68 (d, 2H, 7.5); 3.70 (s, 3H); 2.75 (q, 2H, J = 7.5); 1.14, 3H, J = 7.5). 13C NMR (75 MHz, DMSO-d6): δ 16.4, 28.0, 54.2, 109.3, 113.3, 125.9, 134.9, 150.01, 159.6. Method B. Synthesis of (2-Ethyl-4-methoxy)phenylboronic Acid. a. Preparation of 3-Ethylanisole. To a mixture of 3-ethyl-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
phenol (50 g, 0.4 mol) and K2CO3 (283 g, 2.05 mol) in dry acetone (500 mL) was added methyl iodide (290 g, 2.05 mol). The reaction mixture was transferred to an autoclave and refluxed at 70 °C overnight. The reaction mixture was filtered through a Celite pad, the pad was washed with acetone, and the combined filtrate and washes were concentrated. The product was dissolved in CH2Cl2, filtered, and evaporated to dryness. Yield: 50 g, 90%, as a brown liquid. 1H NMR (400 MHz, CDCl3): δ 7.20-7.28 (m, 1H); 6.74-6.94 (m, 3H); 3.83 (s, 3H); 2.66 (q, 2H, J = 7.2); 1.28 (t, 3H, J=7.7). b. Preparation of (4-Bromo-3-ethyl)anisole. 3-Ethylanisole (50 g, 0.3676 mol) and N-bromosuccinimide (72 g, 0.4 mol) in acetonitrile (1 L) were stirred for 8 h in the dark at room temperature. The reaction mixture was concentrated, keeping the temperature below 40 °C, and the residue obtained was redissolved in CCl4 and filtered. The filtrate was concentrated, and the product was purified by fractional distillation. Yield: 35 g, 43%, as pale-yellow liquid. 1H NMR (300 MHz, CDCl3): δ 7.35-7.45 (m, 1H); 6.80 (d, 1H, J=2.9 Hz); 6.63 (dd, 1H, J=3.0, 8.7 Hz;); 3.80 (s, 3H); 2.74 (q, 2H, J=7.5); 1.23 (t, 3H, J=7.6). c. Preparation of (2-Ethyl-4-methoxy)phenylboronic Acid. The (4-bromo-3-ethyl)anisole was converted to the corresponding boronic acid as described in method A, part c, above. The boronic acid product had 1H and 13C NMR spectra identical with those of the product obtained in method A, part c, above. Synthesis of (S)-2-((9H-Fluoren-9-yloxy)carbonylamino)3-(20 -ethyl-40 -methoxybiphenyl-4-yl)propanoic Acid. a. Preparation of Boc-L-tyrosine O-Triflate Methyl Ester. To a solution of Boc-L-tyrosine methyl ester (85 mmol, 25 g) and 2,6-lutidine (339 mmol, 36.25 g) in CH2Cl2 (200 mL) at -40 °C under N2 was slowly added a solution of triflic anhydride ((169.5 mmol, 47.74 g) in CH2Cl2 (100 mL) over 30 min. The solution was stirred at -40 °C for an additional 2 h, and after this time HPLC analysis indicated that the reaction was complete. The reaction was quenched by addition of water (20 mL). The layers were separated, and the organic layer was washed with 1 N aqueous HCl (3 200 mL), saturated aqueous Na2CO3 (200 mL), water (200 mL), and saturated aqueous NaCl (200 mL). The organic layer was dried over MgSO4, filtered, and concentrated to give the crude product as a red oil. The crude product was purified by silica gel column chromatography (300 g silica gel, 0-50% EtOAc in hexanes as a gradient) to give the desired compound (27 g, 75% yield) as a white solid. 1H NMR (300 MHz, CDCl3): δ 7.10-7.30 (m, 4H); 5.01-5.06 (br m, 1H); 4.55-4.65 (br m, 1H); 3.70 (s, 3H); 3.15-3.23 (m, 1H); 3.01-3.11 (m, 1H); 1.42 (s, 9H). b. Preparation of (S)-2-(tert-Butoxycarbonylamino)-3-(20 -ethyl-40 -methoxybiphenyl-4-yl)propanoic Acid Methyl Ester. Boc-Ltyrosine O-triflate methyl ester (81 g, 0.19 mol) in dry toluene (600 mL) was purged for 10 min with nitrogen. K2CO3 (36 g, 0.26 mol) in water (200 mL) was added, followed by 2-ethyl-4methoxyphenylboronic acid (36 g, 0.2 mol). To the solution were added Pd(PPh3)4 (16.18 g, 0.014 mol), ethanol (200 mL), and THF (400 mL), and the reaction mixture was then heated to 100 °C for 4 h. After this time, the reaction mixture was concentrated under vacuum and the residue was dissolved in CH2Cl2 (1.0 L). The organic layer was washed with 10% aqueous sodium hydroxide, 15% aqueous citric acid, dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography, eluting with 10% EtOAc in petroleum ether. Yield: 50 g, 65%, as a yellow liquid. 1H NMR (300 MHz, CDCl3): δ 7.10-7.30 (m, 5H); 6.86 (d, 1H, J=2.4 Hz); 6.78 (dd, 1H, J=2.5, 8.4 Hz); 4.95-5.05 (br m, 1H); 4.60-4.70 (br m, 1H); 3.85 (s, 3H); 3.71 (s, 3H); 3.05-3.20 (br m, 2H); 2.56 (q, 2H, J = 7.5 Hz); 1.43 (s, 9H); 1.11 (t, 3H, J=7.5 Hz). c. Preparation of (S)-2-(tert-Butoxycarbonylamino)-3-(20 -ethyl-40 -methoxybiphenyl-4-yl)propanoic Acid. To a mixture of (S)2-(tert-butoxycarbonylamino)-3-(20 -ethyl-40 -methoxybiphenyl4-yl)propanoic acid methyl ester (60 g, 0.146 mol) in THF
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(450 mL) and MeOH (85 mL) was added a solution of sodium hydroxide (24 g, 0.58 mol) dissolved in water (85 mL). The reaction mixture was stirred at room temperature overnight. After this time the solution was concentrated and the residue dissolved in water (100 mL) The aqueous layer was washed with Et2O and then was acidified to pH 1 using 20% aqueous citric acid. The aqueous solution was extracted with EtOAc. The organic extracts were combined and washed with saturated aqueous NaCl, dried over Na2SO4, filtered, and concentrated. Yield: 55 g, 94%, as colorless liquid. 1H NMR (300 MHz, DMSO): δ 7.26-7.29 (m, 2H); 7.15-7.20 (m, 2H); 7.04 (d, 1H, J=8.3 Hz); 6.75-6.87 (m, 3H); 4.10-4.18 (br m, 1H); 3.77 (s, 3H); 3.02-3.05 (m, 1H); 2.70-2.85 (m, 1H); 2.50 (q, 2H, J= 7.5 Hz); 1.32 (s, 9H); 1.02 (t, 3H, J=7.5 Hz). d. Preparation of (S)-2-((9H-Fluoren-9-yloxy)carbonylamino)3-(20 -ethyl-40 -methoxybiphenyl-4-yl)propanoic Acid. (S)-2-(tertButoxycarbonylamino)-3-(20 -ethyl-40 -methoxybiphenyl-4-yl)propanoic acid (55 g, 0.138 mol) was dissolved in dry CH2Cl2 (1 L), and dry HCl gas was passed through the reaction mixture at room temperature for 6 h. A precipitate formed which was collected by filtration and then dried under vacuum to give the desired product (46 g, 100%). The resulting free amino acid hydrochloride salt (30 g, 0.089 mol) was dissolved in THF (700 mL), and a solution of NaHCO3 (29 g, 0.358 mol) predissolved in water (240 mL) was added. To the resuling solution was added Fmoc-OSu (30 g, 0.089 mol) portionwise over a period of 30 min. The reaction mixture was stirred overnight at room temperature. The THF was removed under vacuum, and water (2.0 L) was added. The clear solution was washed with Et2O. The aqueous solution was acidified to pH 1 and extracted with EtOAc. The organic layer was washed with water and saturated aqueous NaCl. The EtOAc layer was dried over Na2SO4, filtered, and concentrated to yield 37 g (80%) of product as a pale-yellow solid. LC-MS: (M þ H)þ at m/z 1 522.0; [R]20 D -14.5° (c 1% in DMF). H NMR (400 MHz, DMSO-d6): δ 7.88 (2H, d, J=8.0 Hz); 7.82 (d, 1H); 7.67 (2H, t, J=7.0 Hz); 7.32 (4H, t, J=8.0 Hz); 7.14 (2H, d, J=7.9); 6.99 (1H, d, J=8.4); 6.84 (1H, d, J=2.4); 6.75, (1H, d); 4.10-4.30 (4H, m); 3.77 (3H, s); 3.10-3.20 (1H, m); 2.88-2.98 (1H, m); 2.44 (2H, q, J=7.6); 0.94 (3H, t, J=7.5). 13C NMR (100 MHz, DMSO-d6): δ 14.0, 15.4, 20.7, 25.7, 36.3, 46.6, 55.0, 55.4, 59.7, 65.7, 11.0, 114.0, 120.1, 125.2, 125.3, 127.0,127.6, 128.9, 130.7, 133.4, 136.2, 139.2, 140.7,142.4, 143.7, 143.8, 156.0, 158.6, 170.3, 173.4. Solid Phase Synthesis of Substituted Biphenylalanines,. Method A. 4-(20 ,40 -Dimethoxyphenylaminomethyl)phenoxy (Rink) resin (50 mg, 0.025 mmol) was derivatized with a N-Boc-4iodophenylalanine residue either attached directly to the solid support to synthesize a variety of position 11 biphenylalanine analogues or via coupling to Rink resin already derivatized with an amino acid to synthesize variety of position 10 biphenylalanine analogues. The resulting derivatized Rink resins were weighed into 13 mm 100 mm glass culture tubes with screw caps. Arylboronic acids (0.5 mmol) were dissolved in 0.75 mL of 25% by volume DIEA in N-methylpyrolidinone and added to the resins followed by 0.05 mL of an N-methylpyrolidinone solution containing 1.0 mg of tetrakis(triphenylphospine)palladium(0) catalyst (∼3.5 mol %). The resulting mixtures were blanketed with a stream of nitrogen and the reaction vessels tightly capped and maintained at 85-90 °C for 17-20 h with periodic shaking. The resins were washed with 5 1 mL of N-methylpyrolidinone and 5 1 mL of CH2Cl2 prior to Boc group cleavage as described below. Method B. The reactions were performed as in method A except that a different catalyst was employed. The catalyst solution was prepared by dissolving 9.0 mg of palladium(II) acetate and 56 mg of 2-(dicyclohexylphosphino)biphenyl in 2.0 mL of N-methylpyrolidinone. For 0.025 mmol scale reactions, 0.038 mL (∼3 mol %) of catalyst solution was used.
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Procedures for Cleavage of the Boc Group. Procedure A. The Boc-protected resins prepared as described in method A or B were treated with 0.5 mL of a solution consisting of trimethylsilyl trifluoromethanesulfonate, 2,6-lutidine, and CH2Cl2 (1:1:3 by volume). After three treatments for 1 h each with stirring, the resins were washed with 4 1 mL of CH2Cl2, 3 1 mL of DMP, 3 1 mL of 20% MeOH in DMF, and 4 1 mL CH2Cl2 prior to the next coupling step. Procedure B. The Boc-protected resins prepared as described in method A or B were treated with 1.0 mL of 1 N HCl in anhydrous 1,4-dioxane for 1 h at room temperature with shaking. The resins were washed with 4 1 mL of CH2Cl2, 3 1 mL of 5% DIEA in CH2Cl2 (vol/vol), 3 1 mL of CH2Cl2, and 5 1 mL of N-methylpyrolidinone to provide the free amino resins ready for the next coupling step. Synthesis of Fmoc-(S)-2-fluoro-r-methylphenylalanine. a. Preparation of (R,S)-2-Fluoro-r-methylphenylalanine Hydrochloride. A suspension of N-benzoylalanine (50 g, 0.259 mol) in CH2Cl2 (350 mL) under nitrogen was cooled to 0 °C, and a solution of DCC (58.7 g, 0.28 mol) dissolved in CH2Cl2 (150 mL) was added. The mixture was stirred at 0 °C for 30 min followed by stirring at room temperature for 30 min. The reaction progress was monitored by TLC. Upon disappearance of starting material the reaction mixture was cooled to 0 °C and filtered. The organic layer was concentrated, keeping the temperature below 30 °C. Yield: 44 g as a pale-greenish yellow liquid, which was used in the next step immediately. The oxazolidone from the previous step (44 g, 0.25 mol) was dissolved in DMF (220 mL) under nitrogen. To this solution was added 2-fluorobenzyl bromide (38 g, 0.2 mol). The reaction mixture was cooled to 0 °C, and DIEA (48 g, 0.38 mol) was added dropwise over a period of 30 min. The reaction mixture was stirred at 0 °C for 30 min and at room temperature for 30 min with TLC monitoring. The reaction mixture was quenched with water (500 mL) and extracted with EtOAc (3 200 mL). The combined organic layers were washed with water, saturated aqueous NaCl, dried over Na2SO4, filtered, and concentrated. Yield: 50 g, as a paleyellow liquid. The crude product was taken into the next step without purification. To the benzylated oxazolidone from the previous step (50 g, 0.176 mol) was added 6 N HCl (150 mL) and Et2O (300 mL). The reaction mixture was stirred at room temperature for 1 h. A precipitate formed, and the solid product was collected by filtration and dried under vacuum to yield 50 g of solid product, 60% overall yield for the last three steps. The benzoylated amino acid from the previous step (98 g, 0.42 mol) was dissolved in dioxane (300 mL), and to this solution was added concentrated HCl (700 mL). The reaction mixture was refluxed for 48 h. After removal of the dioxane in vacuo, the aqueous layer was washed with CH2Cl2 and the aqueous layer was concentrated. The residue was triturated with petroleum ether. Yield: 69 g (70%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6): δ 7.30-7.40 (2H, m); 7.12-7.22 (2H, m); 3.38-3.42 (2H, m); 1.46 (3H, s). b. Preparation of N-Trifluoroacetyl-(R,S)-2-fluoro-r-methylphenylalanine. To (R,S)-2-fluoro-R-methylphenylalanine hydrochloride from the previous step (220 g, 0.95 mol) was added TFA (2.2 L) under nitrogen. The solution was cooled to 0 °C. Trifluoroacetic anhydride (500 g, 2.4 mol) was added through an addition funnel dropwise over a period of 30 min, and the reaction mixture was stirred at room temperature overnight. After removal of TFA in vacuo, water (1 L) was added and the mixture was extracted with EtOAc. The combined EtOAc layers were washed with water and saturated aqueous NaCl, dried over Na2SO4, filtered, and concentrated. The product was precipitated with Et2O and collected by filtration. The collected solid was washed with Et2O and then dried under vacuum. Yield: 220 g, 75% as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 7.19-7.21 (1H, m); 7.18-7.13 (3H, m); 3.43-3.48 (1H, m); 3.03-3.08 (1H, m); 1.29 (3H, s).
c. Preparation of N-Fmoc-(S)-2-fluoro-r-methylphenylalanine. To a suspension of N-trifluoroacetyl-(R,S)-2-fluoro-Rmethylphenylalanine (35 g, 0.12 mol) in water (300 mL) was added 1 N aqueous sodium hydroxide until pH 8 was attained. The suspension then became a clear solution. Phosphate buffer (600 mL, pH 7.2) was added to maintain pH 7.2. The mixture was heated to 37 °C. Carboxypeptidase A (60 000 units) was added, and the reaction mixture was stirred overnight. HPLC analysis showed 39% conversion. Then 6 N HCl was added to bring the pH between 3 and 4. Activated charcoal (35 g) was added to the solution, and stirring was continued at room temperature for 15 min. The reaction mixture was filtered through a Celite pad, and the pad was washed with water. The filtrate was acidified to pH 1 using 6 N HCl and extracted with EtOAc and CH2Cl2 to remove unreacted trifluoroacetylamino acid. The aqueous layer was concentrated, and the pH was brought up to 7 using a NaHCO3 solution. THF (300 mL) and Fmoc-OSu (23.6 g, 0.07 mol) were added. The pH was adjusted to 8 by adding additional NaHCO3. The reaction mixture was stirred for 72 h. The solution was acidified with 6 N HCl to pH 2 and extracted with EtOAc. The organic layer was washed with water, dried over Na2SO4, filtered, and concentrated. The product was purified by silica gel column chromatography, eluting with a gradient of using CH2Cl2/EtOAc starting from 100% CH2Cl2 to 10% EtOAc/CH2Cl2. Yield: 8 g, 16%. LC-MS: (M þ H)þ at m/z 420.0;. [R]26 D -40.8° (c 0.634% in DMF). 1H NMR (400 MHz, DMSO-d6): δ 7.90-7.93 (2H, d, m), 7.73-7.76 (2H, m); 7.40-7.44 (3H, m), 7.33-7.36 (2H, m), 7.27-7.29 (1H, m), 7.10-7.14 (1H, m); 7.04-7.12 (1H, m); 6.90-6.95 (1H, m); 4.49-4.53 (1H, m); 4.20-4.30 (2H, m); 3.26-3.37 (1H, m), 3.05-3.10 (1H, m), 1.18 (3H, s). 13C NMR (100 MHz, DMSO-d6): δ 22.3, 32.9, 46.9, 58.5.0, 65.1, 114.8, 115.0, 120.1, 123.5, 123.6, 123.9, 125.1, 125.3, 127.0, 127.6, 128.5, 128.6, 133.0, 140.8, 142.4, 143.8, 143.9, 154.9, 160.0, 162.4, 175.1. Peptide Synthesis. The 11-mer GLP-1 analogues were prepared by solid-phase methods employing N-Fmoc/tert-butyl protection strategy and chemistry. The following side chain protection strategy was employed for standard amino acid residues: Asp(OtBu), Glu(OtBu), His(Trt), Ser(OtBu), Thr(OtBu), and Tyr(OtBu). Solid phase assembly was carried out in a stepwise manner on an Advanced ChemTech MPS 396 multiple peptide synthesizer or on an Applied Biosystems 433A peptide synthesizer using N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU)/DIEA or 1-hydroxy-7-azabenzotriazole (HOAt)/diisopropylcarbodiimide (DIC) coupling chemistry at 0.01-0.25 mmol resin scale (Fmoc Rink or Sieber amide resin). For each coupling cycle, 5-10 equiv of N-Fmoc-amino acid, 20-40 equiv of DIEA, and 5-10 equiv of coupling agent were used. The coupling time was, on average, 1-2 h but was extended to 16 h when coupling a quaternary amino acid in manual mode. Fmoc deprotections were carried out with two treatments of a 15% piperidine in DMF solution, the first for 3 min and the second for an additional 18 min. Peptide Cleavage and Global Deprotection. The target peptides were cleaved/deprotected from their respective peptidyl resins by treatment with a TFA cleavage mixture as follows. For peptide arrays synthesized on the MPS 396 multiple peptide synthesizer at 10-30 μmol/well, a solution of TFA/water/ triisopropylsilane (94:3:3) (1.0 mL) was added to each well in the reactor block, which was then vortexed for 2 h. The TFA solutions from the wells were collected by positive pressure into pretared vials located in a matching 96-vial block on the bottom of the reactor. The resins in the wells were rinsed twice with an additional 0.5 mL of the TFA mixture, and the rinses were combined with the solutions in the vials. These were dried in a SpeedVac (Savant) to yield the crude peptides, typically in >100% yields (20-60 mg). The crude peptides were either washed with ether or more frequently redissolved directly in 2
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mL of DMSO or 50% aqueous acetic acid for purification by preparative HPLC as follows. Preparative HPLC Purification of the Crude Peptides. Preparative HPLC was carried out on either a Waters model 4000 or a Shimadzu model LC-8A liquid chromatograph. Each solution of crude peptide was injected into a YMC S5 ODS (20 mm 100 mm) column and eluted using a linear gradient of MeCN in water, both buffered with 0.1% TFA. A typical gradient used was from 20% to 70% of 0.1% TFA/MeCN in 0.1% TFA/water over 15 min at a flow rate of 14 mL/min with effluent UV detection at 220 nm. The desired product eluted was well separated from impurities, typically after 10-11 min, and was usually collected in a single 10-15 mL fraction on a fraction collector. The desired peptides were obtained as amorphous white powders by lyophilization. HPLC Analysis of the Purified Peptides. Peptide purity was determined by RP-HPLC using the conditions and methods described below. In most cases, the purity was g95%. Exceptions are noted in Table S1 in the Supporting Information. After purification by preparative HPLC, each peptide was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD or LC-10AT analytical HPLC system consisting of a SCL-10A system controller, a SIL-10A autoinjector, and a SPD10AV or SPD-M6A UV/vis detector or a SPD-M10A diode array detector. The column used was a YMC ODS S3 (4.6 mm 50 mm, 3 μm) column or a Phenomenex Luna C18 (4.6 mm 50 mm, 5 μm) column. Elution was performed using one of the following gradients: 0-90% B in A over 8 min, 2.5 mL/min (method A); 20-60% B in A over 8 min, 2.5 mL/min (method B); 20-80% B in A over 8 min, 2.5 mL/min (method C); 10-100% B in A over 8 min, 2.5 mL/min (method D); 10-80% B in A over 8 min, 2.5 mL/min (method E); 10-65% B in A over 8 min, 2.5 mL/min (method F); 5-70% B in A over 8 min, 2.5 mL/min (method G); 30-70% B in A over 8 min, 2.5 mL/min (method H). Mobile phase A was 0.1% TFA/water. Mobile phase B was 0.1% TFA/ acetonitrile. Characterization by Mass Spectrometry. Each peptide was characterized by electrospray mass spectrometry (ES/MS) either in flow injection or in LC-MS mode. Finnigan SSQ7000 single quadrupole mass spectrometers (ThermoFinnigan, San Jose, CA) were used in all analyses in positive and negative ion electrospray modes. Full scan data were acquired over the mass range of 300-2200 amu for a scan time of 1.0 s. The quadrupole was operated at unit resolution. For flow injection analyses, the mass spectrometer was interfaced to a Waters 616 HPLC pump (Waters Corp., Milford, MA) and equipped with an HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland). Samples were injected into a mobile phase containing 50:50 water/acetonitrile with 0.1% ammonium hydroxide. The flow rate for the analyses was 0.42 mL/min, and the injection volume was 6 μL. A ThermoSeparations Constametric 3500 liquid chromatograph (ThermoSeparation Products, San Jose, CA) and HTS PAL autosampler were used for LC-MS analyses. Chromatographic separations were achieved employing a Luna C18, 5 μm column, 2 mm 30 mm (Phenomenex, Torrance, CA). The flow rate for the analyses was 1.0 mL/min and column effluent was split so that the flow into the electrospray interface was 400 μL/min. A linear gradient from 0% to 100% B in A over 4 min was run, where mobile phase A was 98:2 water/acetonitrile with 10 mM ammonium acetate and mobile phase B was 10:90 water/acetonitrile with 10 mM ammonium acetate. The UV response was monitored at 220 nm. The samples were dissolved in 200 μL 50:50 H2O/MeCN (0.05% TFA). The injection volume was 5 μL. In all cases, the experimentally measured molecular weight was within 0.5 Da of the calculated monoisotopic molecular weight (see Supporting Information). cAMP Accumulation Assay. Functional activity was assessed by measuring the ability of the peptides to stimulate intracellular cAMP accumulation in clonal Chinese hamster ovary (CHO) cell lines stably overexpressing either human or mouse GLP-1
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receptors. These cell lines were cultured in F-12 nutrient medium (Ham, Gibco) supplemented with 10% fetal bovine serum and 0.4 mg/mL (human GLP-1R) or 1 mg/mL (mouse GLP-1R) Geneticin at 37 °C in a humidified 5% CO2 atmosphere. Cells were plated at 2 104 cells/well in sterile 96-well white clear bottom Costar plates and cultured overnight before assays. Peptides were serially diluted at half-log concentrations in 100% DMSO and diluted to the desired test concentration in assay buffer (Dulbecco’s phosphate-buffered saline buffer with 0.1 mM 3-isobutyl-1-methylxanthine and 0.05% bovine serum albumin) to give a final concentration of 1% DMSO. To initiate the assay, the medium was aspirated and the cells were treated for 30 min with the diluted agonists (or vehicle) in assay buffer. The cAMP in the cells was measured using the scintillation proximity assay (SPA) method according to the manufacturer’s protocol (Amersham). The counts per minute of 3H-cAMP measured were converted to picomoles of cAMP by interpolating from a cAMP standard curve. The data for the test peptides were normalized and plotted as the percentage of the response stimulated by saturating (10 nM) GLP-1. The data were analyzed by nonlinear regression curve fitting (four-parameter sigmoidal dose-response curve) to determine the EC50, which was defined as the concentration of peptide that produced a half-maximal effect. Each concentration-response was run in duplicate in a given assay, and each peptide was assayed on at least three separate occasions unless otherwise noted. Global fitting to the modified Gaddum/Schild model (GraphPad Prism software, version 4.03) was used to determine the concentration of an antagonist that caused a 2-fold increase in the EC50 value for cAMP accumulation. Intraperitoneal Glucose Tolerance Tests. The protocols for all in vivo studies were approved by the Institutional Animal Care and Use Committee. Male ob/ob mice were purchased at 16 weeks of age (Jackson Laboratories) and were maintained on Teklad 2018 rodent chow (Harlan) until the study was initiated. For the study, 19 week old ob/ob mice were randomized into groups of 6 mice per group based on fed plasma glucose and body weight. After an 18 h overnight fast, mice were weighed and placed in the experimental lab. After 30 min in the environment, the mice were bled via tail tip at -30 min and immediately injected subcutaneously with vehicle (1:1 propylene glycol, 20 mM sodium phosphate, pH 7.4) or the peptides dissolved in vehicle (0.1 mL solution/100 g body weight at a concentration in nmol/mL equivalent to the dose that was to be administered in nmol/kg). At time 0 the mice were bled and then injected intraperitoneally with 50% glucose (2 g/kg) to initiate the glucose tolerance test (IPGTT). The mice were bled 30, 60, 120, and 180 min after the glucose injection. Plasma was prepared from blood samples that had been drawn into potassium EDTA. Plasma glucose concentrations were measured with the Cobas Mira analyzer, and plasma insulin concentrations were determined using the ultrasensitive mouse insulin ELISA kit (Crystal Chem Inc.). Pharmacokinetics Assessment. The pharmacokinetic parameters of peptides 21 and 22 were determined in male beagle dogs (n=3, 14 ( 1 kg). Following an overnight fast, each animal received peptide by subcutaneous injection given near the shoulder blades (67 μg/kg). Each animal received a subcutaneous dose with a 1-week washout between doses. The dosing vehicle for both routes of administration was propylene glycol/ 20 mM sodium phosphate buffer, pH 7.4 (1:1). Serial blood samples were collected in EDTA-containing microcentrifuge tubes at predose, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 24, and 30 h postdose after intravenous administration and at predose, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 24, and 30 h postdose after subcutaneous administration. Approximately 0.3 mL of blood was collected at each time point. Blood samples were immediately centrifuged at 4 °C. The obtained plasma was frozen with dry ice and stored at -20 °C. Plasma drug levels were determined using a LC-MS/ MS assay. Plasma samples were prepared for analysis by
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precipitating plasma proteins with two volumes of acetonitrile containing an internal standard. The samples were vortex mixed, and the precipitated proteins were removed by centrifugation. The resulting supernatants were transferred to a 96-well plate, and an amount of 10 μL was injected for analysis. Samples were prepared with the Packard Multiprobe II and Quadra 96 liquid handling system. The HPLC system consisted of two Shimadzu LC10AD pumps (Columbia, MD) and a CTC PAL autosampler (Leap Technologies, Switzerland). The column used was a YMC Hydrosphere C18 (2.0 mm50 mm, 3 μm) (YMC, Inc., Milford, MA). The column temperature was maintained at 50 °C, and the flow rate was 0.3 mL/min. The mobile phase A consisted of 10 mM ammonium formate and 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The initial mobile phase composition was 5% B and remained at 5% B for 1 min to equilibrate the column. The composition was ramped to 95% B over 2 min and held there for an additional 1 min. The mobile phase was then returned to initial conditions in 1 min. Total analysis time was 5 min. A switching valve was used. The eluents between 0 and 1 min were diverted to the waste. The HPLC was interfaced to a Sciex API 4000 mass spectrometer (Applied Biosystems, Foster City, CA) and was equipped with a TurboIonspray ionization source. Ultrahigh purity nitrogen was used as the nebulizing and turbo gas. The temperature of turbo gas was set at 300 °C, and the interface heater was set at 60 °C. Data acquisition utilized selected reaction monitoring (SRM). Ions representing the (M þ 2H)2þ species for peptides 21 and 22 and for the internal standard were selected in Q1 and were collisionally dissociated with high purity nitrogen at a pressure of 3.5 10-3 torr to form specific product ions which were subsequently monitored by Q3. NMR Spectroscopy. NMR experiments were performed at 600 MHz using 2.8 mM samples of peptide 21 dissolved in 75% DMSO-d6 and 25% H2O (or 25% D2O) at 10 °C. Assignments were obtained from analysis of 2D NOESY, TOCSY, and DQFCOSY spectra. Chemical shifts are referenced to the residual DMSO line at 2.62 ppm. Additional details are presented in Supporting Information.
Acknowledgment. The authors acknowledge Drs. Chet Meyers, John Wetterau, Simeon Taylor, Robert Zahler, and Wesley Cosand for their support of this work. Supporting Information Available: Analytical and spectral characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.
References (1) Wild, S.; Roglic, G.; Green, A.; Sicree, R.; King, H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004, 27, 1047–1053. (2) Holst, J. J.; Orskov, C.; Nielsen, O. V.; Schwartz, T. W. Truncated glucagon-like peptide I, an insulin-releasing hormone from the distal gut. FEBS Lett. 1987, 211, 169–174. (3) Kreymann, B.; Williams, G.; Ghatei, M. A.; Bloom, S. R. Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet 1987, 2, 1300–1304. (4) Mojsov, S.; Weir, G. C.; Habener, J. F. Insulinotropin: glucagonlike peptide I (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J. Clin. Invest. 1987, 79, 616–619. (5) Nauck, M. A.; Heimesaat, M. M.; Behle, K.; Holst, J. J.; Nauck, M. S.; Ritzel, R.; Hufner, M.; Schmiegel, W. H. Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers. J. Clin. Endocrinol. Metab. 2002, 87, 1239–1246. (6) Nauck, M. A.; Niedereichholz, U.; Ettler, R.; Holst, J. J.; Orskov, C.; Ritzel, R.; Schmiegel, W. H. Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans. Am. J. Physiol. 1997, 273, E981–E988.
Article (7) Turton, M. D.; O’Shea, D.; Gunn, I.; Beak, S. A.; Edwards, C. M.; Meeran, K.; Choi, S. J.; Taylor, G. M.; Heath, M. M.; Lambert, P. D.; Wilding, J. P.; Smith, D. M.; Ghatei, M. A.; Herbert, J.; Bloom, S. R. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996, 379, 69–72. (8) Davis, H. R., Jr.; Mullins, D. E.; Pines, J. M.; Hoos, L. M.; France, C. F.; Compton, D. S.; Graziano, M. P.; Sybertz, E. J.; Strader, C. D.; Van Heek, M. Effect of chronic central administration of glucagon-like peptide-1 (7-36) amide on food consumption and body weight in normal and obese rats. Obes. Res. 1998, 6, 147–156. (9) Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel-group study. Lancet 2002, 359, 824–830. (10) Drucker, D. J. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol. Endocrinol. 2003, 17, 161–171. (11) Larsen, J.; Hylleberg, B.; Ng, K.; Damsbo, P. Glucagon-like peptide-1 infusion must be maintained for 24 h/day to obtain acceptable glycemia in type 2 diabetic patients who are poorly controlled on sulphonylurea treatment. Diabetes Care 2001, 24, 1416–1421. (12) Vilsboll, T.; Agerso, H.; Krarup, T.; Holst, J. J. Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects. J. Clin. Endocrinol. Metab. 2003, 88, 220–224. (13) Mentlein, R. Dipeptidyl-peptidase IV (CD26);role in the inactivation of regulatory peptides. Regul. Pept. 1999, 85, 9–24. (14) Holst, J. J. Glucagon-like peptide-1: from extract to agent. The Claude Bernard Lecture, 2005. Diabetologia 2006, 49, 253–260. (15) Eng, J.; Kleinman, W. A.; Singh, L.; Singh, G.; Raufman, J. P. Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. J. Biol. Chem. 1992, 267, 7402–7405. (16) Buse, J. B.; Henry, R. R.; Han, J.; Kim, D. D.; Fineman, M. S.; Baron, A. D. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with type 2 diabetes. Diabetes Care 2004, 27, 2628–2635. (17) Green, B. D.; Flatt, P. R. Incretin hormone mimetics and analogues in diabetes therapeutics. Best Pract. Res., Clin. Endocrinol. Metab. 2007, 21, 497–516. (18) Chen, D.; Liao, J.; Li, N.; Zhou, C.; Liu, Q.; Wang, G.; Zhang, R.; Zhang, S.; Lin, L.; Chen, K.; Xie, X.; Nan, F.; Young, A. A.; Wang, M. W. A nonpeptidic agonist of glucagon-like peptide 1 receptors with efficacy in diabetic db/db mice. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 943–948. (19) Knudsen, L. B.; Kiel, D.; Teng, M.; Behrens, C.; Bhumralkar, D.; Kodra, J. T.; Holst, J. J.; Jeppesen, C. B.; Johnson, M. D.; de Jong, J. C.; Jorgensen, A. S.; Kercher, T.; Kostrowicki, J.; Madsen, P.; Olesen, P. H.; Petersen, J. S.; Poulsen, F.; Sidelmann, U. G.; Sturis, J.; Truesdale, L.; May, J.; Lau, J. Small-molecule agonists for the glucagon-like peptide 1 receptor. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 937–942. (20) Rink, H. Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin. Tetrahedron Lett. 1987, 28, 3787–3790. (21) King, D. S.; Fields, C. G.; Fields, G. B. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int. J. Pept. Protein Res. 1990, 36, 255–266. (22) Gefel, D.; Hendrick, G. K.; Mojsov, S.; Habener, J.; Weir, G. C. Glucagon-like peptide-I analogs: effects on insulin secretion and adenosine 30 ,50 -monophosphate formation. Endocrinology 1990, 126, 2164–2168. (23) Suzuki, S.; Kawai, K.; Ohashi, S.; Mukai, H.; Yamashita, K. Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and
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glucagon release from the isolated perfused rat pancreas. Endocrinology 1989, 125, 3109–3114. Adelhorst, K.; Hedegaard, B. B.; Knudsen, L. B.; Kirk, O. Structure-activity studies of glucagon-like peptide-1. J. Biol. Chem. 1994, 269, 6275–6278. Gallwitz, B.; Witt, M.; Paetzold, G.; Morys-Wortmann, C.; Zimmermann, B.; Eckart, K.; Folsch, U. R.; Schmidt, W. E. Structure/activity characterization of glucagon-like peptide-1. Eur. J. Biochem. 1994, 225, 1151–1156. Siegel, E. G.; Gallwitz, B.; Scharf, G.; Mentlein, R.; MorysWortmann, C.; Folsch, U. R.; Schrezenmeir, J.; Drescher, K.; Schmidt, W. E. Biological activity of GLP-1-analogues with N-terminal modifications. Regul. Pept. 1999, 79, 93–102. Deacon, C. F.; Knudsen, L. B.; Madsen, K.; Wiberg, F. C.; Jacobsen, O.; Holst, J. J. Dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1 which have extended metabolic stability and improved biological activity. Diabetologia 1998, 41, 271–278. Ritzel, U.; Leonhardt, U.; Ottleben, M.; Ruhmann, A.; Eckart, K.; Spiess, J.; Ramadori, G. A synthetic glucagon-like peptide-1 analog with improved plasma stability. J. Endocrinol. 1998, 159, 93–102. Knudsen, L. B.; Nielsen, P. F.; Huusfeldt, P. O.; Johansen, N. L.; Madsen, K.; Pedersen, F. Z.; Thogersen, H.; Wilken, M.; Agerso, H. Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration. J. Med. Chem. 2000, 43, 1664–1669. Madsen, K.; Knudsen, L. B.; Agersoe, H.; Nielsen, P. F.; Thogersen, H.; Wilken, M.; Johansen, N. L. Structure-activity and protraction relationship of long-acting glucagon-like peptide-1 derivatives: importance of fatty acid length, polarity, and bulkiness. J. Med. Chem. 2007, 50, 6126–6132. Parker, J. C.; Andrews, K. M.; Rescek, D. M.; Massefski, W., Jr.; Andrews, G. C.; Contillo, L. G.; Stevenson, R. W.; Singleton, D. H.; Suleske, R. T. Structure-function analysis of a series of glucagon-like peptide-1 analogs. J. Pept. Res. 1998, 52, 398–409. Raufman, J. P.; Singh, L.; Singh, G.; Eng, J. Truncated glucagonlike peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4. J. Biol. Chem. 1992, 267, 21432– 21437. Thornton, K.; Gorenstein, D. G. Structure of glucagon-like peptide (7-36) amide in a dodecylphosphocholine micelle as determined by 2D NMR. Biochemistry 1994, 33, 3532–3539. Murage, E. N.; Schroeder, J. C.; Beinborn, M.; Ahn, J. M. Search for alpha-helical propensity in the receptor-bound conformation of glucagon-like peptide-1. Bioorg. Med. Chem. 2008, 16, 10106– 10112. Agerso, H.; Jensen, L. B.; Elbrond, B.; Rolan, P.; Zdravkovic, M. The pharmacokinetics, pharmacodynamics, safety and tolerability of NN2211, a new long-acting GLP-1 derivative, in healthy men. Diabetologia 2002, 45, 195–202. Elbrond, B.; Jakobsen, G.; Larsen, S.; Agerso, H.; Jensen, L. B.; Rolan, P.; Sturis, J.; Hatorp, V.; Zdravkovic, M. Pharmacokinetics, pharmacodynamics, safety, and tolerability of a single-dose of NN2211, a long-acting glucagon-like peptide 1 derivative, in healthy male subjects. Diabetes Care 2002, 25, 1398–1404. Fineman, M. S.; Shen, L. Z.; Taylor, K.; Kim, D. D.; Baron, A. D. Effectiveness of progressive dose-escalation of exenatide (exendin4) in reducing dose-limiting side effects in subjects with type 2 diabetes. Diabetes Metab. Res. Rev. 2004, 20, 411–417. Ritzel, R.; Orskov, C.; Holst, J. J.; Nauck, M. A. Pharmacokinetic, insulinotropic, and glucagonostatic properties of GLP-1 [7-36 amide] after subcutaneous injection in healthy volunteers. Doseresponse-relationships. Diabetologia 1995, 38, 720–725.