Intermediate for synthesis of FAPI-QS
Fibroblast activation protein (FAP). UAMC-1110 (and its derivatives) is a highly potent, specific, and irreversible FAP inhibitor. It displays low activity against related dipeptidyl peptidases (DPPIV, DPP9, DPPII) and prolyl oligopeptidase (PREP). The NH2 derivative serves as a scaffold for conjugating the FAP-binding motif to chelators (e.g., DOTA, NODAGA) for radiolabeling or to fluorescent dyes for imaging. The parent molecule has a Ki in the low nanomolar range.

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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No specific in vitro activity data reported for the NH2 derivative itself. The parent compound, UAMC-1110, has demonstrated potent and specific inhibition of FAP at nanomolar concentrations. It is a gold-standard chemical probe for studying the role of FAP in cancer-associated fibroblasts (CAFs) and tumor microenvironment. The derivative is used to create conjugates (FAPI) that retain this high-affinity binding to FAP-expressing CAFs.

No specific in vivo data reported for the NH2 derivative. UAMC1110-NH2 is a research intermediate. However, the conjugates it is used to create (e.g., FAPI-QS radiotracers) are highly effective in vivo. When labeled with a positron-emitting radioisotope (e.g., 68Ga, 18F), these FAPIs can be used for PET/CT imaging of FAP-expressing tumors, including many cancers (breast, lung, pancreatic, etc.). This allows visualization of the tumor microenvironment and is a very powerful clinical diagnostic tool.

UAMC1110-NH2 is a derivative of a potent and specific FAP inhibitor. A typical procedure for developing a radiotracer involves first conjugating UAMC1110-NH2 with a bifunctional chelator (e.g., DOTAGA, NODAGA-NHS) via its reactive primary amine. The resulting conjugate (e.g., DOTAGA-UAMC1110) is then purified by HPLC. The labeling of the conjugate with a radiometal (e.g., 68Ga3+ or 177Lu3+) is achieved by incubating the conjugate with the radioisotope in a buffer (e.g., HEPES, sodium acetate) at 80-100degC for 10-30 minutes. The final radiotracer is then formulated for injection. Binding assays are performed on FAP-positive cells or purified FAP protein.

UAMC1110-NH2 is not used directly, but the resulting FAPI conjugates are tested. For cell binding and inhibition studies, a cancer-associated fibroblast (CAF) cell line (e.g., HEK293 cells overexpressing FAP or patient-derived CAFs) is seeded in 12-well plates. Cells are treated with UAMC-1110 or a FAPI conjugate (1 pM-1 uM) for 1 hour. FAP enzyme activity is assessed by adding a specific fluorogenic substrate (e.g., Z-Gly-Pro-7-amido-4-methylcoumarin, Z-GP-AMC). Fluorescence (excitation 380 nm, emission 460 nm) is read at 37degC for 30-60 minutes. The IC50 is calculated from a dose-response curve.

For a 68Ga-labeled FAPI radiotracer derived from UAMC1110-NH2, a typical protocol involves intravenous (i.v.) injection of the tracer (e.g., 5-10 mCi, 100-200 uL) via the tail vein into 8-12 week old female mice bearing subcutaneous xenografts of FAP-positive human cancer cells (e.g., HT-1080 fibrosarcoma or patient-derived xenograft models). At 60-120 minutes post-injection, animals are euthanized, and positron emission tomography/computed tomography (PET/CT) images are acquired. Organs and tumors are dissected, weighed, and the radioactive uptake (percentage of injected dose per gram of tissue, %ID/g) is measured using a gamma counter. Biodistribution is the primary endpoint.

Not applicable for the linker. The PET imaging tracers derived from UAMC1110-NH2 are designed for optimal imaging pharmacokinetics. When labeled with 68Ga (t½ = 68 min), the tracer has a short half-life. It is rapidly cleared from the blood via the kidneys (renal clearance), resulting in very high target-to-background ratios within 1-2 hours post-injection. This is ideal for same-day imaging. For therapy, longer-lived isotopes like 177Lu (t½ = 6.7 days) would be used, which changes the PK profile.

UAMC1110-NH2 is a chemical derivative of a potent FAP inhibitor. No specific toxicity data is reported for this intermediate. The parent compound, UAMC-1110, is a research chemical with low toxicity at sub-micromolar concentrations. The final FAPI radiotracers are typically well-tolerated in both animal and human studies at imaging doses. The primary risk is the inherent risk of the radiometal (e.g., irradiation for 68Ga, which is low, or significant for 177Lu). All necessary radiochemical safety precautions must be followed when handling these materials. The toxicity profile of the unlabeled conjugate is compound-specific.

[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. doi: 10.1186/s41181-020-00102-z.

UAMC1110-NH2 is a key building block for the synthesis of FAPI (fibroblast activation protein inhibitor)-based radiopharmaceuticals (e.g., FAPI-QS, FAPI-04, FAPI-46). These have revolutionized nuclear oncology imaging, allowing for the visualization of the tumor stroma. This is complementary to FDG-PET, which visualizes the cancer cells themselves. FAP is highly expressed in the cancer-associated fibroblasts (CAFs) of over 90% of epithelial carcinomas but is low in normal adult tissues. Molecular formula: C21H23F2N5O3; molecular weight: 431.44.

C21H23F2N5O3

431.435831308365

431.18

C, 58.46; H, 5.37; F, 8.81; N, 16.23; O, 11.12

2758337-19-6

2990021-73-1 (NH2-UAMC1110 TFA)

156499857

Solid powder

1.4

2

8

8

31

698

1

C1[C@H](N(CC1(F)F)C(=O)CNC(=O)C2=C3C=C(C=CC3=NC=C2)OCCCCN)C#N

WDZAGMQBKLWFSD-AWEZNQCLSA-N

InChI=1S/C21H23F2N5O3/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/h3-5,7,9,14H,1-2,6,8,10,12-13,24H2,(H,27,30)/t14-/m0/s1

(S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide

UAMC1110-NH2; UAMC 1110-NH2; UAMC-1110-NH2; NH2-UAMC1110; SCHEMBL23531732; NH2-UAMC1110; NH2-UAMC-1110; Amino-UAMC1110;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)