Intermediate for synthesis of FAPI-QS

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]

[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]

C23H24F5N5O5

545.46

545.1697

2990021-73-1

NH2-UAMC1110;2758337-19-6

163197094

Off-white to yellow solid powder

3

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.)