FAP (IC50 = 3.2 nM); PREP (IC50 = 1.8 μM)
In vitro activity: SP-13786 (formerly known as FAP-IN-1) is a novel, potent and highly selective inhibitor of fibroblast activation protein (FAP) with IC50 of 3.2 nM; it also inhibits prolyl oligopeptidase (PREP) with an IC50 of 1.8 μM. Fibroblast activation protein (FAP) is a serine protease related to dipeptidyl peptidase IV (DPPIV). It has been convincingly linked to multiple disease states involving remodeling of the extracellular matrix. FAP inhibition is investigated as a therapeutic option for several of these diseases, with most attention so far devoted to oncology applications. The log D values, plasma stabilities, and microsomal stabilities of SP-13786 were found to be highly satisfactory. Pharmacokinetic evaluation in mice of SP-13786 demonstrated high oral bioavailability, plasma half-life, and the potential to selectively and completely inhibit FAP in vivo.

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Kinase Assay: SP-13786 (formerly known as FAP-IN-1) is a novel, potent and highly selective inhibitor of fibroblast activation protein (FAP) with IC50 of 3.2 nM; it also inhibits prolyl oligopeptidase (PREP) with an IC50 of 1.8 μM.

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Cell Assay: FAP-IN-1 is also found to have better FAP/PREP selectivity and a very proficient ligand efficiency of 0.34.

In vitro activity: SP-13786 (formerly known as FAP-IN-1) is a novel, potent and highly selective inhibitor of fibroblast activation protein (FAP) with IC50 of 3.2 nM; it also inhibits prolyl oligopeptidase (PREP) with an IC50 of 1.8 μM. Fibroblast activation protein (FAP) is a serine protease related to dipeptidyl peptidase IV (DPPIV). It has been convincingly linked to multiple disease states involving remodeling of the extracellular matrix. FAP inhibition is investigated as a therapeutic option for several of these diseases, with most attention so far devoted to oncology applications. The log D values, plasma stabilities, and microsomal stabilities of SP-13786 were found to be highly satisfactory. Pharmacokinetic evaluation in mice of SP-13786 demonstrated high oral bioavailability, plasma half-life, and the potential to selectively and completely inhibit FAP in vivo.

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Kinase Assay: SP-13786 (formerly known as FAP-IN-1) is a novel, potent and highly selective inhibitor of fibroblast activation protein (FAP) with IC50 of 3.2 nM; it also inhibits prolyl oligopeptidase (PREP) with an IC50 of 1.8 μM.

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Cell Assay: FAP-IN-1 is also found to have better FAP/PREP selectivity and a very proficient ligand efficiency of 0.34.

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UAMC1110 exhibits potent inhibitory activity against FAP with an IC₅₀ of 3 nM and high selectivity over the related prolyl peptidase PREP (IC₅₀ > 1.8 µM, SI > 562.5). It shows no significant inhibition against DPPIV, DPP9, or DPP2 at concentrations up to 100 µM.
The compound demonstrates high kinetic solubility (>200 µM) and a log D value of 1.0.
It displays satisfactory stability in mouse (85% unchanged after 6 h) and rat (95% unchanged after 6 h) plasma.
It also shows high stability in rat hepatic microsomes (94% unchanged after 6 h).
No cytotoxicity was observed against MRC-5 human embryonic lung fibroblasts at concentrations up to 64 µM.[1]

FAP-IN-1 is the most extensive and prolonged inhibitior of FAP in the PK studies. No tight binding behavior is observed, and the inhibitor proves to bind reversibly to FAP. Pharmacokinetic evaluation in mice of FAP-IN-1 demonstrates high oral bioavailability, plasma half-life, and the potential to selectively and completely inhibit FAP in vivo

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Following a single oral administration (20 mg/kg) in male Wistar rats, UAMC1110 achieved a Cₘₐₓ of 14.6 µg/mL at Tₘₐₓ of 0.33 h, with an elimination half-life (T₁/₂) of 3.4 h and an oral bioavailability of 74%.
Ex vivo analysis of plasma samples from treated rats showed that a single oral dose (20 mg/kg) of UAMC1110 resulted in >85% inhibition of plasma FAP activity at all time points measured (5 min to 24 h), indicating complete or near-complete and prolonged target engagement in vivo.[1]

Reversibility of the Probes Based on a Dialysis Experiment[2]
Recombinant human FAP (rhFAP) was incubated for 15 min at 37°C with a concentration of probe that was predicted to inhibit around 90% of FAP’s activity (5: 1.08 nM; 6: 2.50 nM; 7: 1.35 nM; UAMC1110: 0.77 nM diluted in FAP assay buffer: 50 mM Tris-HCl pH 8.0, 140 mM NaCl, and 1 mg/ml BSA). As a solvent control, rhFAP was incubated with 0.0002% DMSO. After 15 min of incubation, FAP activity was determined as published by Bracke et al. (2019). Subsequently, the samples were dialyzed at 4°C against FAP assay buffer (using a 10 kDa cut-off Slide-A-Lyzer MINI dialysis device. Buffer (14 ml) was exchanged after 3 h, 6 h, 24 h, 3 days, and 7 days, and after each of these time points, a FAP activity measurement was performed. For the UAMC1110 parent compound, FAP activity was only measured on days 3 and 7.

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The inhibitory potency (IC₅₀) of compounds against FAP, PREP, DPPIV, DPP9, and DPP2 was determined using spectrophotometric assays with specific chromogenic substrates.
FAP activity was assayed using Ala-Pro-p-nitroanilide (2 mM) as substrate in 50 mM Tris, 1 M NaCl, 1 mg/mL bovine serum albumin, pH 7.4. Recombinant murine FAP was used.
PREP activity was assayed using Z-Gly-Pro-p-nitroanilide (0.25 mM) as substrate in 50 mM Tris, pH 7.5. Recombinant human PREP was used.
DPPIV activity was assayed using Gly-Pro-p-nitroanilide (100 µM) as substrate in 50 mM Tris, pH 8.3. Enzyme was purified from human seminal plasma.
DPPII activity was assayed using Lys-Ala-p-nitroanilide (1 mM) as substrate in 50 mM citrate-phosphate, pH 5.5. Enzyme was purified from human seminal plasma.
DPP9 activity was assayed using Ala-Pro-p-nitroanilide (300 µM) as substrate in 50 mM Tris, 1 mg/mL bovine serum albumin, pH 7.4. Enzyme was purified from bovine testes.
Substrate concentrations were chosen around the Kₘ values. Inhibitors were pre-incubated with enzyme for 10 min at 37°C before adding substrate. The increase in absorbance at 405 nm was monitored. IC₅₀ values were calculated from dose-response curves.[1]

Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP's function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for in situ FAP activity staining in patient-derived cryosections of urothelial tumors.Front Chem. 2021 Apr 14;9:640566.

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Cytotoxicity was evaluated on MRC-5SV2 human embryonic diploid fibroblasts. Cells were cultured in MEM medium supplemented with L-glutamine, NaHCO₃, and 5% inactivated fetal calf serum at 37°C under 5% CO₂.
Cells were exposed to the test compound for a specified duration. Cell viability was assessed using the MTT assay. The highest concentration tested was 64 µM.[1]

20 mg/kg; p.o.; 5 mg/kg. i.v.
Rats: The PK parameters are determined for inhibitors 4, 5, 60 (FAP-IN-1), and 61 in rats. Six male rats are treated for each inhibitor tested, three of which received the compound via a single intravenous (iv) administration at 5 mg/kg. The other three animals are dosed per os (po) at 20 mg/kg. Blood samples are collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, and 24 h after administration. Inhibitor concentrations are determined using UPLC MS/MS, and pharmacokinetic parameters are calculated using standard algorithms

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For pharmacokinetic studies, male Wistar rats (~250 g) were used. For each compound, six rats were divided into two groups (n=3 per group).
One group received a single intravenous (iv) administration of the compound formulated in PEG₂₀₀ at a dose of 5 mg/kg.
The other group received a single oral (po) administration (gavage) of the compound formulated in PEG₂₀₀ at a dose of 20 mg/kg.
Blood samples were collected via tail vein or cardiac puncture at time points: 0.083, 0.25, 0.5, 1, 2, 4, 6, and 24 h after administration.
Plasma was separated and stored at -20°C until analysis. Plasma concentrations of the inhibitor were determined using UPLC-MS/MS. Pharmacokinetic parameters were calculated using standard non-compartmental analysis.[1]
For ex vivo FAP inhibition assessment, residual FAP activity was measured in the plasma samples collected during the PK study, using the substrate Z-Gly-Pro-AMC under conditions excluding soluble PREP activity. Activity was compared to vehicle-treated controls.[1]

Oral bioavailability in rats = 51% T1/2 = 3.2 h hr UAMC1110 showed high kinetic solubility (>200 µM). The measured log D value was 1.0. It exhibited good stability in both mouse plasma (85% unchanged after 6 hours) and rat plasma (95% unchanged after 6 hours). It showed high stability in rat liver microsomes (94% unchanged after 6 hours). In rats, after a single intravenous injection (5 mg/kg), its Cₘₐₓ was 11.8 µg/mL, AUC was 23.4 µg·h/mL, clearance (Cl) was 2.83 mL/min, volume of distribution (Vz) was 0.43 L, and half-life (T₁/₂) was 1.74 h. After a single oral administration (20 mg/kg), the time to peak concentration (Tₘₐₓ) was 0.33 hours, the plasma concentration (Cₘₐₓ) was 14.6 µg/mL, the area under the curve (AUC) was 76.7 µg·h/mL, the clearance (Cl) was 1.55 mL/min, the volume of distribution (Vz) was 0.34 L, the half-life (T₁/₂) was 3.4 hours, and the relative oral bioavailability was 74%. [1]

No signs of cytotoxicity were observed in MRC-5 cells at the highest tested concentration (64 µM). [1]
In in vivo rat pharmacokinetic studies, all animals treated with UAMC1110 (intravenous and oral) showed no signs of toxicity during the observation period or at necropsy, in stark contrast to compound 4, which caused death. [1]

[1]. Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-cyanopyrrolidine inhibitors of fibroblast activation protein (FAP). J Med Chem. 2014 Apr 10;57(7):3053-74.

Fibroblast activating protein (FAP) is a serine protease associated with dipeptidyl peptidase IV (DPPIV). It has been shown to be closely associated with a variety of disease states involving extracellular matrix remodeling. FAP inhibitors are being investigated as potential strategies for treating a variety of such diseases, with current research primarily focused on oncology applications. We previously discovered that the N-4-quinolinyl-Gly-(2S)-cyanoproline backbone may be a potential starting structure for highly efficient and selective FAP inhibitors. In this study, we explored the structure-activity relationship surrounding this core backbone in detail. We report well-optimized compounds that exhibit low nanomolar inhibitory activity and high selectivity against the associated dipeptidyl peptidases (DPPs) DPPIV, DPP9, DPPII, and prolyl oligopeptidase (PREP). The selected compounds showed very satisfactory log D values, plasma stability, and microsomal stability. Pharmacokinetic evaluations in mice indicated that the selected inhibitors have high oral bioavailability, short plasma half-life, and the potential for selective and complete inhibition of FAP in vivo. [1]

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Fibroblast activating protein (FAP) is a proline-selective protease belonging to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME), particularly in cancer-associated fibroblasts, the main cellular component of the tumor stroma. The exact role of its enzymatic activity in the TME remains unclear. Therefore, tools that can selectively and activity-based visualize FAP in the TME are helpful in revealing the function of FAP. We describe the synthesis, biochemical characterization, and application of three different active probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP inhibitor UAMC1110. UAMC1110 is a molecule we developed in-house and is considered to be the most potent and selective FAP inhibitor currently available. We demonstrate that these three probes have sub-nanomolar FAP affinity and significant selectivity for relevant S9 family members. Furthermore, we found that fluorescent Cy3- and Cy5-labeled probes can selectively detect FAP in the cellular environment, making these chemical probes ideal for further biological research. In addition, we provide a proof-of-concept for in situ FAP activity staining in frozen sections of patient-derived urothelial tumors. [2] UAMC1110 is a (4-quinolineyl)glycyl-2-cyanopyrrolidine derivative, specifically a compound having a 4,4-difluorinated pyrrolidine ring at the P1 position. It is one of the most potent and selective FAP inhibitors reported in this study. The compound binds reversibly to FAP and does not exhibit slow, tight binding kinetics. The observed prolonged in vitro FAP inhibition is primarily attributed to its favorable pharmacokinetic half-life. The authors highlight the structural similarity of this series of compounds and note that unexpected toxicity of the parent compound 4 was observed in rats for reasons that are unclear, while no such toxicity was observed in UAMC1110. [1]

C₁₇H₁₄F₂N₄O₂

344.32

344.108

C, 59.30; H, 4.10; F, 11.04; N, 16.27; O, 9.29

1448440-52-5

1448440-52-5;

71621488

Typically exists as White to off-white solid at room temperature

1.4±0.1 g/cm3

676.4±55.0 °C at 760 mmHg

362.9±31.5 °C

0.0±2.1 mmHg at 25°C

1.621

0.22

1

6

3

25

588

1

FC1(CN(C(CNC(C2C=CN=C3C=CC=CC=23)=O)=O)[C@H](C#N)C1)F

PUOOCZVRHBHJRS-NSHDSACASA-N

InChI=1S/C17H14F2N4O2/c18-17(19)7-11(8-20)23(10-17)15(24)9-22-16(25)13-5-6-21-14-4-2-1-3-12(13)14/h1-6,11H,7,9-10H2,(H,22,25)/t11-/m0/s1

N-[2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl]quinoline-4-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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