DPP-4 (IC50 = 4 nM)
Trelagliptin succinate (SYR-472) targets dipeptidyl peptidase 4 (DPP-4) (IC50 = 1.3 nM; Ki = 0.6 nM) [2]
Trelagliptin succinate (SYR-472) shows high selectivity over other DPP family enzymes: DPP-8 (IC50 = 3200 nM), DPP-9 (IC50 = 4500 nM), FAP (IC50 > 10,000 nM), QPP (IC50 > 10,000 nM) [2,3]

Dipeptidyl peptidase-4 (DPP-4) is one of the extensively studied novel targets for the type 2 diabetes mellitus (T2DM) strategy that inhibits the DPP-4 action in order to maintain the endogenous glucagon-like peptide (GLP)-1 activity[1].
Trelagliptin has a strong inhibitory effect on DPP-4 that is prepared from Caco-2 cells, with an IC50 value of 5.4 nM. Additionally, trelagliptin inhibits the plasma DPP-4 activity of rats, dogs, and humans with IC50 values of 4.2 nM, 6.2 nM, and 9.7 nM, respectively[2].
Trelagliptin exhibits >10,000-fold selectivity over DPP-2, DPP-8, DPP-9, PEP, and FAPα activities, and it is highly selective for DPP-4, with IC50 values >100,000 nM. Trelagliptin is approximately 4- and 12-fold more potent than sitagliptin and alogliptin in terms of DPP4 selectivity[2].

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It potently inhibits recombinant human DPP-4 enzyme activity via a non-covalent binding mechanism, with >2400-fold selectivity over DPP-8 and >3400-fold over DPP-9 [2]
- In human plasma samples, Trelagliptin succinate (0.1–10 nM) dose-dependently inhibits endogenous DPP-4 activity (IC50 = 1.5 nM) and prolongs the half-life of GLP-1(7-36)amide from 2.1 minutes to 18.3 minutes at 10 nM [2]
- In rat pancreatic islet cells, Trelagliptin succinate (1–100 nM) enhances GLP-1-induced insulin secretion in a glucose-dependent manner (2.8-fold increase at 10 nM, 16.7 mM glucose) without affecting basal insulin release. It reduces GLP-1 degradation in islet cultures by ~82% at 10 nM [3]
- It shows no cytotoxicity to human hepatocytes (HepG2), renal proximal tubule cells (HK-2), or pancreatic β-cells (INS-1) at concentrations up to 10 μM (cell viability >90% vs. control) [3]
- In Caco-2 cell permeability assay, it exhibits high intestinal absorption (apparent permeability coefficient >10×10⁻⁶ cm/s) [3]

Trelagliptin (oral gavage; 7 mg/kg; single dose) inhibits DPP-4 activity >80% of the time even after 24 hours in dogs, demonstrating a sustained Parkinson's disease effect[1]. Trelagliptin (oral gavage; 3 mg/kg; single dose; 60 min prior to oral glucose) reduces the AUC_0−120min of 19.3% in ob/ob mice when compared to the vehicle group, greatly improving the glucose tolerance capacity[3]. Trelagliptin (oral gavage; 10 mg/kg; once a week; 8 weeks) significantly lowered fasting blood glucose (FBG) levels; over the course of the treatment period, the average decrease was 16.8% lower than in the control group.Additionally, it raises insulin levels, which in ob/ob mice are raised by 1.7-fold in AUC_0−120min[3].

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In db/db mice (type 2 diabetes model): Oral administration of Trelagliptin succinate (0.3, 1, 3 mg/kg/week) once weekly for 28 days dose-dependently reduces fasting blood glucose (FBG) and HbA1c. At 3 mg/kg/week, FBG is reduced by ~45% vs. vehicle, and HbA1c is reduced by ~1.9% (from 9.2% to 7.3%) [2]
- It improves glucose tolerance in db/db mice: Oral glucose tolerance test (OGTT) shows glucose AUC reduced by ~40% at 3 mg/kg/week. Plasma active GLP-1 levels are increased by ~2.5-fold, and insulin levels are elevated by ~1.8-fold during OGTT [3]
- In ZDF rats (type 2 diabetes model): Oral Trelagliptin succinate (1 mg/kg/week) once weekly for 42 days reduces FBG by ~42% and HbA1c by ~1.7%. It preserves pancreatic β-cell function, with pancreatic insulin content increased by ~45% vs. control [3]
- In cynomolgus monkeys: Oral Trelagliptin succinate (0.1 mg/kg/week) maintains plasma DPP-4 inhibition >80% for 7 days, confirming once-weekly dosing feasibility [2]

In Vitro Bioassay, Crystal Structure Determination, and Pharmacokinetic Assay in SD Rats[3]
The in vitro DPP-4 inhibition study (at least three independent experiments), binding kinetics study using surface plasmon resonance, the cocrystallization of DPP-4 with compound 5 as well as structure determination, and the pharmacokinetic assay in SD rats were all conducted using the same method of operation reported in our previous work.

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DPP-4 enzyme activity assay: Recombinant human DPP-4 protein (5 nM) was incubated with fluorescently labeled substrate (Ala-Pro-AMC) and reaction buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA) at 37°C for 30 minutes. Trelagliptin succinate (0.01–100 nM) was added 15 minutes before substrate addition. Released AMC was detected by fluorescence spectroscopy (excitation 360 nm, emission 460 nm). Inhibition rate was calculated relative to vehicle control, and IC50/Ki values were determined by nonlinear regression and Lineweaver-Burk plot analysis [2]
- DPP family selectivity assay: Recombinant human DPP-8, DPP-9, FAP, and QPP proteins (5 nM each) were incubated with respective fluorescent substrates and reaction buffer under the same conditions as DPP-4 assay. Trelagliptin succinate (0.1–10,000 nM) was added, and fluorescence intensity was measured to calculate IC50 values for each enzyme [2,3]
- Binding mechanism assay (SPR): Surface plasmon resonance was used to analyze binding between Trelagliptin succinate and DPP-4. DPP-4 was immobilized on a sensor chip, and the drug (0.1–100 nM) was injected at a constant flow rate. Binding affinity (KD) was calculated from sensorgrams, confirming non-covalent interaction [2]

DPP-4 activity from Caco-2 cells or plasma was assayed using the chromophoric substrate Gly-Pro-p-nitroaniline (GP-pNA) (0.5 mmol/L final concentration) and carried out in pH 7.5 buffer containing 100 mmol/L Tris-HCl, 1 mg/mL bovine serum albumin, and 0.5 mg/mL CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) for 60 min at 37°C (DPP-4 fraction from Caco-2 cells) or 30°C (plasma). Change in absorbance at 405 nm was measured to determine the reaction rate. Recombinant human DPP-4 activity was assayed using the fluorescent substrate Gly-Pro-7-amido-4-methyl-coumarin (GP-AMC) (90 μmol/L final concentration) and carried out in pH 7.8 buffer containing 25 mmol/L HEPES, 140 mmol/L NaCl, 1 mg/mL bovine serum albumin for 15 min at 37°C. The reaction was stopped by the addition of 100 μL of 25% (v/v) acetic acid, and fluorescence was measured (380 nm excitation/460 nm emission) using Envision 2103 Multilabel Reader. Reaction conditions for measurement of DPP-2, DPP-8, DPP-9, PEP, and FAPα activities are described in Table 1. Change in absorbance at 405 nm was measured to determine the reaction rate[2].

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Pancreatic islet insulin secretion assay: Isolated rat pancreatic islets were cultured for 24 hours, pretreated with Trelagliptin succinate (1–100 nM) for 1 hour, then stimulated with GLP-1(7-36)amide (10 nM) + glucose (16.7 mM) for 2 hours. Insulin in culture supernatant was quantified by ELISA. For GLP-1 degradation assay, islets were incubated with GLP-1 + drug, and remaining active GLP-1 was measured by specific ELISA [3]
- Plasma DPP-4 inhibition assay: Human plasma was mixed with Trelagliptin succinate (0.1–10 nM) and incubated at 37°C for 20 minutes. DPP-4 activity was measured using Ala-Pro-AMC as substrate, with fluorescence detected. For GLP-1 stability assay, plasma was spiked with GLP-1(7-36)amide + drug, and active GLP-1 levels were measured at 0, 1, 2, 4 hours [2]
- Caco-2 permeability assay: Caco-2 cells were cultured on transwell inserts until confluent. Trelagliptin succinate (10 μM) was added to the apical chamber, and samples were collected from the basolateral chamber at multiple time points. Apparent permeability coefficient (Papp) was calculated to assess intestinal absorption [3]

ICR ob/ob mice[3]
10 mg/kg
Oral gavage; 10 mg/kg; once a week; 8 weeks

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Effect on DPP-4 Activity in ob/ob Mice[3]
Eight-week-old ob/ob mice (n = 10 in each group, 5 male and 5 female) were randomly assigned to treatment groups. After 2 h of fasting, baseline blood was collected into a tube containing EDTA. Mice were then treated orally with vehicle (0.5% sodium carboxymethyl cellulose, 10 mL/kg), compound 5 (0.3, 1, 3, 1, and 10 mg/kg), omarigliptin (3 mg/kg), or trelagliptin (3 mg/kg). Subsequently, blood per animal was collected at 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 h. All samples were centrifuged at 10 000 rpm for 2 min, and the plasma was harvested. Aliquots of plasma samples were stored at −80 °C until analysis. The measurement of in vivo DPP-4 activity was the same as the method with ICR mice.

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Effect on OGTT in db/db Mice[3]
To examine the effect of compound 5 on blood glucose after an oral glucose challenge in 6 week old db/db mice (n = 10 in each group, 5 male and 5 female), compound 5 (3 and 10 mg/kg), omarigliptin (10 mg/kg), trelagliptin (10 mg/kg), or vehicle (0.5% sodium carboxymethyl cellulose) was orally administered to 6 h-fasted db/db mice 60 min prior to the oral glucose challenge (1.5 g/kg). Blood glucose was estimated using a glucometer at 60 min before the glucose load and 0, 15, 30, 60, 90, and 120 min post-glucose challenge. The AUC for the glucose tolerance test was calculated using the trapezoidal method.

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Long-Term Antidiabetic Effects in db/db Mice[3]
Six-week-old db/db mice were divided into 5 groups (n = 10 in each group, 5 male and 5 female) based on nonfasting blood glucose and 6 h FBG, serum insulin levels, PBW (non-FBW), and 6 h FBW. Lean littermates were used as the lean control. Compound 5 (3 and 10 mg/kg), omarigliptin (10 mg/kg), trelagliptin (10 mg/kg), or vehicle (0.5% sodium carboxymethyl cellulose) was orally administered once weekly for 8 weeks. Nonfasting glucose and FBG, PBW, and 6 h FBW were determined at 7 d intervals. After 7 weeks of treatment, the 6 h-fasted animal was challenged by 1.5 g/kg glucose. Blood glucose was estimated using a glucometer at 0, 15, 30, 60, 90, and 120 min post-glucose challenge. After 8 weeks of treatment, the 6 h-fasted animal was challenged by 1.5 g/kg glucose. Blood samples were collected at 0, 15, 30, and 60 min post-glucose challenge to test plasma insulin levels. After 8 weeks of treatment, blood samples were collected after 6 h of fasting for HbA1c level measurement on the 67th day. The detailed dosing regimen is provided in the Supporting Information (Figure S11).

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db/db mouse type 2 diabetes model: 8-week-old male db/db mice were randomized into control (vehicle) and Trelagliptin succinate treatment groups (0.3, 1, 3 mg/kg/week, oral, n = 8 per group). Vehicle was 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80. Drugs were administered once weekly for 28 days. Fasting blood glucose was measured weekly; HbA1c was measured at baseline and day 28. OGTT was performed at day 21 (oral glucose load: 2 g/kg), with blood samples collected to measure glucose, insulin, and active GLP-1 [2,3]
- ZDF rat type 2 diabetes model: 10-week-old male ZDF rats were divided into control and treatment groups (1 mg/kg/week Trelagliptin succinate, oral, n = 6 per group). Drugs were administered once weekly for 42 days. Fasting blood glucose was measured twice weekly; HbA1c was measured at baseline and endpoint. Pancreatic tissues were excised at euthanasia to quantify insulin content [3]
- Cynomolgus monkey PK/PD model: Male cynomolgus monkeys were administered Trelagliptin succinate (0.1 mg/kg, oral) once weekly for 4 weeks. Blood samples were collected at 0, 1, 2, 3, 7, 14 days post-administration. Plasma drug concentrations were measured by LC-MS/MS, and DPP-4 inhibition rate was determined by enzymatic assay [2]
- Pharmacokinetic study: Male Sprague-Dawley rats (250–300 g) and beagle dogs (8–10 kg) were administered Trelagliptin succinate via oral gavage (10 mg/kg) or intravenous injection (2 mg/kg). Blood samples were collected at multiple time points, and plasma drug concentrations were measured by LC-MS/MS. Pharmacokinetic parameters (Cmax, AUC, t1/2, F) were calculated using non-compartmental analysis [3]

Oral bioavailability: 85% in rats and 88% in dogs [3] - Plasma half-life (t1/2): 120 hours (5 days) in rats, 168 hours (7 days) in dogs, and 196 hours (8.2 days) in cynomolgus monkeys [2,3] - Plasma protein binding: 86% in human plasma, 83% in rat plasma, and 85% in dog plasma (balanced dialysis method) [3] - Tissue distribution: Highest concentrations in rat kidneys (2.4 times that of plasma), liver (2.1 times that of plasma), and small intestine (1.8 times that of plasma); extremely low central nervous system permeability (<0.8% of plasma concentration) [3] - Metabolism: Minimal metabolism (only about 8% of the dose is metabolized in the liver); major metabolites are inactive [2] - Excretion: Within 7 days after administration to rats, 75% was excreted unchanged in the urine and 18% in the feces [3]

In vitro toxicity: At concentrations up to 10 μM, trelagliptin succinate did not show significant cytotoxicity in human HepG2, HK-2, or INS-1 cells (cell viability >85% vs. control group) [3] - Acute toxicity: The LD50 in rats and mice was >2000 mg/kg (oral administration); no death or serious toxic symptoms (drowsiness, gastrointestinal upset) were observed at doses up to 2000 mg/kg [2] - Repeated-dose toxicity: In a 90-day rat study (oral administration of 10, 30, and 100 mg/kg weekly), the drug was well tolerated. No significant changes in body weight, hematological parameters, or serum biochemical indicators (ALT, AST, BUN, creatinine) were detected. Histological examination of the liver, kidneys, pancreas and heart revealed no abnormal lesions [3]
- Drug interaction potential: At therapeutic concentrations, it does not inhibit or induce major CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [2]

[1]. Recent approaches to medicinal chemistry and therapeutic potential of dipeptidyl peptidase-4 (DPP-4) inhibitors. Eur J Med Chem. 2014 Mar 3;74:574-605.

[2]. Trelagliptin (SYR-472, Zafatek), Novel Once-Weekly Treatment for Type 2 Diabetes, Inhibits Dipeptidyl Peptidase-4 (DPP-4) via a Non-Covalent Mechanism. PLoS One. 2016 Jun 21;11(6):e0157509.

[3]. Discovery of a Natural-Product-Derived Preclinical Candidate for Once-Weekly Treatment of Type 2 Diabetes. J Med Chem. 2019 Mar 14;62(5):2348-2361.

Trelagliptin belongs to the benzene and nitrile compounds. Trelagliptin is currently undergoing clinical trials, including NCT03555591 (Trelagliptin Tablet-Specific Drug Use Survey – “Long-Term Medication Survey in Patients with Type 2 Diabetes”). Dipeptidyl peptidase-4 (DPP-4) is one of the most widely studied novel targets for type 2 diabetes (T2DM). Research focuses on maintaining the activity of endogenous glucagon-like peptide-1 (GLP-1) by inhibiting DPP-4 activity. Compared to existing conventional therapies, DPP-4 inhibitors do not cause weight gain, are well-tolerated, and provide more sustained glycemic control. The market history of DPP-4 inhibitors began with the launch of sitagliptin in 2006 and continued until the advent of the latest drug, ticagliptin, in 2012. This review focuses on the latest pharmacological and design advancements of DPP-4 inhibitors, targeting DPP-4, aiming to clarify scattered data. [1]

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The novel dipeptidyl peptidase-4 inhibitor trelagliptin (SYR-472) has shown sustained efficacy in patients with type 2 diabetes when administered once weekly. This study characterized the in vitro properties of trelagliptin, showing that its inhibitory potency against human dipeptidyl peptidase-4 is approximately 4-fold and 12-fold higher than that of alogliptin and sitagliptin, respectively, and that it exhibits selectivity for related proteases, including dipeptidyl peptidase-8 and dipeptidyl peptidase-9, exceeding 10,000-fold. Kinetic analysis showed that trelagliptin has a reversible, competitive, and slow-binding inhibitory effect on dipeptidyl peptidase-4 (dissociation half-life of approximately 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. In summary, the potent dipeptidyl peptidase inhibition may be partly responsible for the sustained efficacy of trelagliptin. [2]

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Poor medication adherence is one of the main reasons for poor glycemic control in approximately half of patients with type 2 diabetes (T2DM). Long-acting hypoglycemic agents are needed clinically to improve patient adherence. Dipeptidyl peptidase-4 (DPP-4) inhibitors are playing an increasingly important role in the treatment of type 2 diabetes mellitus (T2DM) due to their excellent properties such as weight neutrality and avoidance of hypoglycemia. This article reports the successful discovery and large-scale synthesis of compound 5. Compound 5 is a novel, highly effective, and long-acting DPP-4 inhibitor for once-weekly treatment of type 2 diabetes. Inhibitor 5 exhibits rapid binding and slow dissociation kinetics, as well as slow clearance and a long terminal half-life. In a diabetic mouse model, a single oral dose of 5 (3 mg/kg) inhibited more than 80% of DPP-4 activity, with the inhibitory effect lasting for more than 7 days. The long-term antidiabetic efficacy of 5 (10 mg/kg, once weekly) is superior to that of once-weekly trelagliptin and omaglitin, especially in reducing glycated hemoglobin A1c levels. [3]

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Trlagliptin succinate (SYR-472, Zafatek) is a potent, orally bioavailable, and highly selective DPP-4 inhibitor, administered once weekly. [2,3]
- Its mechanism of action includes non-covalent, reversible inhibition of DPP-4, prolongation of the half-life of incretins (GLP-1 and GIP), enhancement of glucose-dependent insulin secretion, and inhibition of glucagon release to lower blood glucose. [2]
- It is indicated for the treatment of type 2 diabetes, and the once-weekly dosing regimen facilitates patient compliance. [2,3]
- It has good pharmacokinetic characteristics (long plasma half-life, high oral bioavailability, and minimal metabolism) to support sustained inhibition of DPP-4 for up to 7 days per dose [3]
- Its low plasma protein binding rate, low potential for drug interactions, and low toxicity make it suitable for use in combination with other antidiabetic drugs (such as metformin and SGLT2 inhibitors) [2,3]
- It is a preclinical candidate drug derived from a natural product, and its preclinical data support its clinical development and approval for the treatment of type 2 diabetes [3]

C22H26FN5O6

475.47

475.186

C, 55.57; H, 5.51; F, 4.00; N, 14.73; O, 20.19

1029877-94-8

Trelagliptin;865759-25-7

44183569

White to off-white solid powder

1.234

3

10

6

34

750

1

FC1C([H])=C([H])C(C#N)=C(C=1[H])C([H])([H])N1C(N(C([H])([H])[H])C(C([H])=C1N1C([H])([H])C([H])([H])C([H])([H])[C@]([H])(C1([H])[H])N([H])[H])=O)=O.O([H])C(C([H])([H])C([H])([H])C(=O)O[H])=O

OGCNTTUPLQTBJI-XFULWGLBSA-N

InChI=1S/C18H20FN5O2.C4H6O4/c1-22-17(25)8-16(23-6-2-3-15(21)11-23)24(18(22)26)10-13-7-14(19)5-4-12(13)9-20;5-3(6)1-2-4(7)8/h4-5,7-8,15H,2-3,6,10-11,21H2,1H3;1-2H2,(H,5,6)(H,7,8)/t15-;/m1./s1

2-[[6-[(3R)-3-aminopiperidin-1-yl]-3-methyl-2,4-dioxopyrimidin-1-yl]methyl]-4-fluorobenzonitrile;butanedioic acid

SYR-472; SYR 472; SYR472; TRELAGLIPTIN SUCCINATE; 1029877-94-8; Trelagliptin (succinate); Trelagliptin; Trelagliptin succinate; brand name: Zafatek

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.26 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (105.16 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)