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

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.)