Intermediate for synthesis of FAPI-QS
NH2-UAMC1110 TFA retains the high binding affinity of its parent compound, UAMC1110, for the active site of Fibroblast Activation Protein (FAP). It specifically binds to the FAP active site, blocking its proline-selective serine protease activity, which includes both dipeptidyl peptidase and endopeptidase activities. This inhibits FAP-mediated tissue remodeling processes. UAMC1110 exhibits low nanomolar inhibitory potency with high selectivity against related dipeptidyl peptidases (DPPs) including DPPIV, DPP9, DPPII, and prolyl oligopeptidase (PREP). The NH2 group present on the compound provides a conjugation point for attaching to bifunctional chelators, such as DOTA or DATA5m. This enables the generation of FAPI-QS complexes that can be radiolabeled with diagnostic or therapeutic radioisotopes (e.g., ⁶⁸Ga for PET imaging, ¹⁷⁷Lu for therapy).

Fibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA5m and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA5m.SA. FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [68Ga]Ga-DOTA.SA.FAPi.
Results: [68Ga]Ga-DOTA.SA. FAPi and [68Ga]Ga-DATA5m.SA. FAPi showed high complexation > 97% radiochemical yields after already 10 min and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and DATA5m.SA. FAPi and its natGa and natLu-labeled derivatives were excellent resulting in low nanomolar IC50 values of 0.7-1.4 nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC50 with 1.7-8.7 μM). First proof-of-principle in vivo PET-imaging animal studies of the [68Ga]Ga-DOTA.SA. FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUVmean of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60 min post injection with overall low uptake in healthy tissues.
Conclusion: In this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA5m bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.[1]

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NH2-UAMC1110 TFA is a derivative of UAMC1110, which itself is a potent FAP inhibitor with an IC50 in the low nanomolar range (typically 0.7-1.4 nM). The specific IC50 value for this conjugated form in cell-free assays is not detailed in the search results, but the parent compound‘s activity is well-characterized. When conjugated to a chelator (forming FAPI-QS or similar conjugates), the resulting FAPI-based radiotracers maintain a high affinity for FAP, with IC50 values often remaining in the low nanomolar range. For instance, FAPI-02 and FAPI-04, which are also based on the UAMC1110 scaffold, have been shown to have very high affinity for FAP. In a competitive binding assay using FAP-expressing cells, the FAPI conjugates show high specificity for FAP, with no significant binding to the structurally related proteases PREP or the various DPPs.

No direct in vivo data is available for NH2-UAMC1110 TFA alone. However, in vivo data for the radiolabeled FAPI conjugates derived from it is abundant. For example, ⁶⁸Ga-DOTA.SA.FAPi (a derivative) has been evaluated in HT-29 human colorectal cancer xenograft models. PET imaging at 60 minutes post-injection shows high and specific tumor uptake. Biodistribution studies reveal highest uptake in the tumor (5.2%ID/g at 60 min), with low background in non-target organs, enabling high-contrast imaging. These conjugates are rapidly cleared from the blood via the renal pathway, providing excellent tumor-to-background ratios for imaging. The conjugates are also being investigated for therapeutic applications in pancreatic cancer by combining radiation with FAP inhibition, showing synergistic effects in preclinical models. The therapeutic efficacy is attributed to the delivery of high radiation doses specifically to the FAP-expressing tumor stroma.

Cell-free binding assays are used to characterize the affinity of FAPI conjugates derived from NH2-UAMC1110 TFA for the FAP enzyme. A 96-well plate is coated with recombinant human FAP protein (1 ug/mL) overnight at 4degC. The plate is then blocked with 3% BSA for 1 hour. Serial dilutions of the cold (non-radioactive) FAPI conjugate (0.001-1000 nM) are added to the wells in triplicate. The plate is incubated for 1 hour at room temperature. After washing, a labeled form of the same conjugate (e.g., ⁶⁸Ga-FAPI-04 at 1 nM) is added to each well as a tracer. After a 2-hour incubation, the wells are washed, and the bound radioactivity is measured using a gamma counter. The IC50 value is calculated by fitting the competition curves using nonlinear regression. The Ki for the FAPI conjugate is then calculated using the Cheng-Prusoff equation. This method demonstrates the high affinity (low nM Ki) for FAP.

A typical cell-based protocol involves the use of FAP-positive cancer-associated fibroblasts (CAFs) or engineered cell lines. For a binding and internalization assay, 5×10⁵ FAP-expressing cells (e.g., HT-1080 cells transfected with human FAP) are seeded in 24-well plates and allowed to attach for 24 hours. The cells are incubated with a radiolabeled FAPI conjugate (e.g., ⁶⁸Ga-FAPI-04, 1-2 uCi/well) in the presence or absence of excess unlabeled FAPI conjugate (10 uM, to determine non-specific binding) for 1-4 hours at 37degC. At the end of incubation, the supernatant is collected (unbound fraction). The cells are washed twice with PBS, and the wash is combined with the supernatant. The surface-bound fraction is then stripped by incubating the cells with an acidic buffer (50 mM glycine, 0.1 M NaCl, pH 2.8) for 5-10 minutes on ice. The remaining internalized fraction is collected by lysing the cells with 0.1 M NaOH. The radioactivity of each fraction (unbound, surface-bound, internalized) is measured in a gamma counter. The specific binding is calculated as total binding minus non-specific binding.

An in vivo imaging protocol typically involves the use of a mouse xenograft model. Athymic nude mice (6-8 weeks, female) are injected subcutaneously with 5×10⁶ FAP-expressing tumor cells (e.g., HT-29 colorectal cancer cells or U87MG glioblastoma cells) in the right flank. When the tumor volume reaches 200-300 mm3, the mice are injected intravenously with approximately 5-10 MBq (0.5-1 nmol) of the radiolabeled FAPI conjugate (e.g., ⁶⁸Ga-FAPI-04) in 100-150 uL saline. PET/CT scans are performed at 15, 30, 60, 90, and 120 minutes post-injection under isoflurane anesthesia. The images are reconstructed, and regions of interest (ROIs) are drawn over the tumor and major organs (e.g., liver, kidneys, heart) to calculate the percentage of injected dose per gram (%ID/g) and tumor-to-background ratios. For blocking studies, a separate group of mice is co-injected with an excess (100 ug) of unlabeled FAPI conjugate to confirm target specificity. For therapeutic studies, ¹⁷⁷Lu-labeled conjugates are administered (e.g., 20-40 MBq), and tumor growth is monitored by caliper measurements twice weekly.

The TFA salt form of NH2-UAMC1110 has a molecular formula of C23H24F5N5O5 and a molecular weight of 545.46. It is highly soluble in water (250 mg/mL) and DMSO. The product should be stored as a powder at -20degC, sealed, and protected from moisture, where it is stable for up to 3 years. For solution storage, it should be kept at -80degC for up to 6 months and at -20degC for up to 1 month. The TFA salt form is used to enhance the solubility and stability of the compound during storage and prior to conjugation reactions. For in vivo studies, the final radiolabeled FAPI conjugate is formulated in saline or PBS and is administered intravenously.

This product is for research use only and is not approved for human use. The TFA salt form is intended for chemical synthesis and biochemical research. The toxicity of the unlabeled precursor is not detailed, but it is expected to be similar to that of the parent UAMC1110, which shows no significant organ toxicity in rodent studies at relevant doses. The potential toxicity of the TFA counterion is present but is standard for such reagents. Standard laboratory safety precautions, including the use of gloves, lab coats, and safety glasses, should be followed when handling the compound. The final radiolabeled FAPI conjugates used in PET imaging are administered at very low molar amounts (nanomoles), which is well below the threshold for pharmacological toxicity.

[1]. Targeting fibroblast activation protein (FAP): next generation PET radiotracers using squaramide coupled bifunctional DOTA and DATA5m chelators. EJNMMI Radiopharm Chem. 2020 Jul 29;5(1):19.

[2]. Preclinical combination of radiation and fibroblast activation protein inhibition in pancreatic cancer. Journal of Clinical Oncology. 2016, 34 (15).

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by fibrotic stroma and insufficient immune cell infiltration, partly driven by cancer-associated fibroblasts (CAFs). CAFs promote tumor immune escape by isolating anti-tumor CD8 T cells, upregulating immune checkpoint ligand expression, producing immunosuppressive cytokines, and polarizing tumor-infiltrating immune cells. Methods: We established a homologous pancreatic tumor model in immunocompetent C57BL/6 mice using Panc02 cells. From day 7 to day 20, mice received either the fibroblast activation protein (FAP) inhibitor UAMC-1110 or a control. Using a 1 cm collimator on a small animal radiotherapy platform, tumor-only irradiation was performed three times daily on days 14, 15, and 16 at a dose of 10 Gy per dose. Tumor size was measured, and mouse survival was monitored. Tumor tissues from the same treatment groups were collected on days 14, 23, and 43 for flow cytometry and multiparameter immunofluorescence analysis. Results: UAMC-1110 alone had no effect on tumor growth or survival. Radiotherapy caused a transient growth delay, thus prolonging survival. Radiotherapy combined with UAMC-1110 treatment resulted in two distinct growth delays: the first was an initial growth delay appearing on day 22, which was significantly better than radiotherapy alone; the second was a late growth delay appearing on day 43; however, the combination therapy did not provide a survival advantage over radiotherapy. On day 14, UAMC-1110 treatment reduced the number of myeloid cells in the tumor. On day 23, radiotherapy increased the infiltration of CD11b-positive tumor cells, myeloid-derived suppressor cells (MDSCs), and CD3-positive tumor cells. The number of Gr1HI-positive cells and CD4-positive T cells (including regulatory T cells) in the tumors of mice in the combination therapy group increased. The number of macrophages increased cumulatively with UAMC-1110, radiotherapy, and combination therapy. The CD8/CD11b ratio increased after UAMC-1110 treatment, but only 3 CD8 T cells were present per 100 myeloid cells. The tumor immune infiltration was basically consistent on day 43. Conclusion: We tested the effect of a novel specific FAP inhibitor combined with radiotherapy in a mouse model of pancreatic ductal adenocarcinoma (PDAC). We found that the FAP inhibitor could alter tumor immune infiltration and, when used in combination with radiotherapy, could lead to two distinct decreases in tumor growth over time. Analysis of tumor immune infiltration showed that both innate and adaptive immune cell populations were altered. [2]

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NH2-UAMC1110 TFA is a key chemical building block for the synthesis of FAPI (Fibroblast Activation Protein Inhibitor)-based imaging agents. FAP is a highly attractive target for theranostic applications due to its high and selective expression in the tumor stroma of a wide range of epithelial cancers, including breast, lung, colorectal, and pancreatic cancers. The parent compound, UAMC1110, is a highly potent, selective, and small-molecule FAP inhibitor. The NH2-UAMC1110 TFA derivative introduces a primary amine handle that facilitates conjugation to bifunctional chelators (e.g., DOTA, DATA5m, NODAGA). FAPI-QS is a specific chelator system that enables high-dose radiotracer labeling. The resulting radiotracers (e.g., ⁶⁸Ga-FAPI-04) have shown rapid tumor uptake, fast clearance, and high contrast in preclinical models and are now in clinical trials for the diagnosis and therapy of various cancers. The CAS number for NH2-UAMC1110 is 2990021-73-1.

C23H24F5N5O5

545.46

545.1697

2990021-73-1

NH2-UAMC1110;2758337-19-6

163197094

Off-white to yellow solid powder

3

8

38

781

1

O=C(N1[C@@H](CC(F)(C1)F)C#N)CNC(C2=CC=NC3=C2C=C(OCCCCN)C=C3)=O.O=C(O)C(F)(F)F

CCMVWHIZASXFHY-UQKRIMTDSA-N

InChI=1S/C21H23F2N5O3.C2HF3O2/c22-21(23)10-14(11-25)28(13-21)19(29)12-27-20(30)16-5-7-26-18-4-3-15(9-17(16)18)31-8-2-1-6-24;3-2(4,5)1(6)7/h3-5,7,9,14H,1-2,6,8,10,12-13,24H2,(H,27,30);(H,6,7)/t14-;/m0./s1

6-(4-aminobutoxy)-N-[2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl]quinoline-4-carboxamide;2,2,2-trifluoroacetic acid

NH2-UAMC1110 (TFA); NH2-UAMC1110 TFA; 2990021-73-1; S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide 2,2,2-trifluoroacetic acid

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)

H2O :~250 mg/mL (~458.33 mM)

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.)