Chemokine receptor CCR10 (antagonist) [1]
.

Inhibition of CCL27-dependent Ca²⁺ Flux: In CHO-K cells stably transfected with human CCR10 and aequorin, the initial screening hit compound 1 inhibited CCL27-dependent Ca²⁺ flux with an IC50 of 690 nM. Optimization led to cut-22, which is approximately 300-fold more potent than compound 1 in this assay. Specifically, cut-22 (racemate) showed a pIC50 of 8.7 ± 0.2 in the FLIPR Ca²⁺ flux assay using CHO-K cells stimulated with CCL27. The active enantiomer, eut-22, had a pIC50 of 9.0 [8.9, 9.1] in the same assay [1]
.
Inhibition of CCL28-dependent Ca²⁺ Flux: cut-22 also inhibited Ca²⁺ flux stimulated by the other known CCR10 ligand, CCL28. In CHO-K cells, the pIC50 for cut-22 against CCL28-stimulated Ca²⁺ flux was 7.9 ± 0.3 (FLIPR assay) and 7.9 ± 0.1 (Aequorin assay). For eut-22, the pIC50 was 8.9 (FLIPR) and 9.0 [8.9, 9.1] (Aequorin) [1]
.
Inhibition of CCL27-dependent Chemotaxis: In Ba/F3 cells stably transfected with human CCR10, compound 1 inhibited CCL27-dependent chemotaxis with an IC50 of 53 nM. cut-22 demonstrated potent antagonism in this functional assay with a pIC50 of 9.0 ± 0.3. The active enantiomer eut-22 showed a pIC50 of 7.6 [7.4, 7.9] for murine CCR10 in a similar chemotaxis assay [1]
.
Competition Binding Assay: cut-22 competitively and reversibly binds to CCR10. In an immunochemical binding assay using HEK cell membrane preparations and an Fc-CCL27 fusion protein as a probe, compound 14 (a close analog) had a binding pIC50 of 8.5. While the exact value for cut-22 is not provided in this specific table, its activity in functional assays is consistent with direct receptor binding [1]
.
GTP Binding Assay: cut-22 affects CCR10's coupling with G-protein. In a GTP-Eu binding assay using HEK cell membrane preparations stimulated with CCL27, cut-22 had a pIC50 of 8.0 [1]
.
Inhibition of CCL27-dependent cAMP Production: cut-22 inhibits CCL27-dependent cAMP production. In HEK cells transfected with CCR10 and stimulated with CCL27, cut-22 showed a pIC50 of 7.6 in the cAMP assay [1]
.
Selectivity: cut-22 is highly selective for CCR10. No meaningful binding or activity was observed against a panel of 29 GPCRs, including six other chemokine receptors (data referenced in Supplementary Material) [1]
.
Structure-Activity Relationship (SAR) for R¹ Group: The nitro group on the imidazole of the initial hit (compound 1, IC50 690 nM) was found to be important but replaceable. The unsubstituted imidazole (compound 2) was less potent. Replacing the nitro with cyano (compound 3) or chloro (compound 4) maintained or improved potency. Further exploration led to the discovery that cyanopyrrole (compound 14, human CCR10 pIC50 7.7±0.2) was a highly effective replacement, eliminating the potential toxicity concerns associated with the nitroimidazole [1]
.
Structure-Activity Relationship (SAR) for R² Group (Sulfonamide): The aniline sulfonamide in the initial hit (compound 1) presented potential toxicity liabilities. While anilines (e.g., compounds 16, 17, 18) were potent, they were replaced with indoles, which have similar hydrogen-bond donating properties. This led to the discovery of the 1H-indol-4-yl sulfonamide in cut-22, which showed significantly improved potency (pIC50 8.7±0.1 for rac-22) compared to similar anilines and eliminated the aniline liability. The indole NH is crucial, as the N-methyl indole analog (23) was ~70-fold less potent [1]
.
Structure-Activity Relationship (SAR) for R³ Group (Amide): The 4-methylpiperidine amide was identified as optimal. Variations in the piperidine 4-substituent (e.g., larger groups like in 27, 28 or polar groups like in 29), the position of the methyl group on the ring (30-33), or changes to the ring size/homologation (34-38) all resulted in decreased potency compared to the 4-methylpiperidine analog [1]
.
Stereospecificity: CCR10 antagonism is highly stereospecific. The homophenylalanine analogs R-10 and S-10 showed a strong preference for one enantiomer. This was also observed for the enantiomers of cut-22, where eut-22 (pIC50 9.0) is the active enantiomer and dis-22 (pIC50 5.5±0.1) is significantly less active [1]
.

In sensitized mice, BI-6901 (ip; 100 mg/kg; bid.) simulates an anti-inflammatory response to DNFB-stimulated ear edema. In the same model, the illness levels shown with BI-6901 matched the therapeutic levels (60–85%) seen with anti-CCL27 antibodies [1].

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Efficacy in Murine DNFB Contact Hypersensitivity Model: The active enantiomer, eut-22, demonstrated efficacy in a mouse model of contact hypersensitivity. When administered intraperitoneally at 100 mg/kg twice daily (0 and 8 hours), eut-22 significantly inhibited ear swelling in Balb-C mice sensitized and challenged with DNFB (2,4-dinitrofluorobenzene). The effect was dose-dependent. The inactive enantiomer, dis-22, showed no activity in the same model, confirming that the efficacy is due to specific CCR10 antagonism. The level of efficacy was similar to that previously reported for an anti-CCL27 antibody (60-85%) [1]
.

GTP-Binding Assay (GTP-Eu): This assay was used to confirm that compounds affect CCR10 coupling with G-protein. HEK cell membranes prepared from cells expressing human CCR10 were used. Membranes were incubated with test compound, the agonist CCL27, and a non-hydrolyzable, europium-labeled GTP analog (GTP-Eu). The binding of GTP-Eu, which reflects G-protein activation, was measured over time using time-resolved fluorescence (TRF). The pIC50 value for inhibition of GTP binding by cut-22 was determined [1]
.
Competition Binding Assay (Fc-CCL27): A binding assay was performed to demonstrate direct and competitive interaction with CCR10. HEK cell membrane preparations containing human CCR10 were used. A fusion protein of Fc and the chemokine CCL27 (Fc-CCL27) was used as a probe. The ability of test compounds to compete with Fc-CCL27 for binding to CCR10 was measured immunochemically. The pIC50 for compound 14 in this assay was determined [1]
.

Ca²⁺ Flux Assay (FLIPR and Aequorin): The primary screening and characterization assay used CHO-K cells stably transfected with human CCR10 and the bioluminescent protein aequorin. For the FLIPR (Fluorescent Imaging Plate Reader) format, cells were loaded with a calcium-sensitive fluorescent dye. Cells were pre-incubated with varying concentrations of test compounds and then stimulated with an agonist (either CCL27 or CCL28). The transient increase in intracellular Ca²⁺, which is a measure of receptor activation, was measured by either fluorescence (FLIPR) or aequorin-generated chemiluminescence. The IC50 values for inhibition of this Ca²⁺ flux were calculated [1]
.
Chemotaxis Assay: This functional assay was performed using Ba/F3 cells stably transfected with human or murine CCR10. Cells were placed in the upper chamber of a Transwell plate. Test compounds were added to the upper chamber, and the chemokine CCL27 was added to the lower chamber as a chemoattractant. After an incubation period, the number of cells that had migrated to the lower chamber was counted. The IC50 for inhibition of CCL27-dependent chemotaxis by test compounds was determined [1]
.
cAMP Assay: This assay was performed in HEK cells transfected with human CCR10. CCR10 activation by CCL27 leads to inhibition of adenylyl cyclase and a decrease in cAMP production. Cells were pre-incubated with test compounds, then stimulated with CCL27 in the presence of forskolin (to elevate baseline cAMP). The intracellular cAMP levels were measured, and the ability of test compounds to reverse the CCL27-mediated inhibition of cAMP production (i.e., to increase cAMP levels) was quantified as a pIC50 [1]
.

Animal/Disease Models: DNFB model of contact hypersensitivity in Balb-C mice.
Doses: 100 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: It has an inhibitory effect on the inflammatory response stimulated by DNFB in sensitized Balb-c mice.

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Murine DNFB Contact Hypersensitivity Model:** Female Balb-C mice were sensitized by topical application of a DNFB solution to the shaved abdomen on two consecutive days. Five days after the initial sensitization, baseline ear thickness was measured. Mice were then challenged by topical application of a DNFB solution to the right ear. Test compounds (eut-22 and dis-22) were formulated in a vehicle (30% cremophore) and administered via intraperitoneal (ip) injection at a dose of 100 mg/kg at two time points: immediately after the DNFB challenge (time 0) and again 8 hours later. A positive control group (Cyclosporine A, 30 mg/kg, route not specified in the protocol part of the text) and a vehicle control group were included. Ear swelling, the measure of the inflammatory response, was quantified 24 hours after the DNFB challenge using a caliper [1]
.

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Murine DNFB Contact Hypersensitivity Model: Female Balb-C mice were sensitized by topical application of a DNFB solution to the shaved abdomen on two consecutive days. Five days after the initial sensitization, baseline ear thickness was measured. Mice were then challenged by topical application of a DNFB solution to the right ear. Test compounds (eut-22 and dis-22) were formulated in a vehicle (30% cremophore) and administered via intraperitoneal (ip) injection at a dose of 100 mg/kg at two time points: immediately after the DNFB challenge (time 0) and again 8 hours later. A positive control group (Cyclosporine A, 30 mg/kg, route not specified in the protocol part of the text) and a vehicle control group were included. Ear swelling, the measure of the inflammatory response, was quantified 24 hours after the DNFB challenge using a caliper [1]
.

Murine Metabolic Stability: cut-22 (and by extension its enantiomers) exhibited high clearance in mice. In murine liver microsome assays, the half-life (t½) was less than 3 minutes, corresponding to a predicted hepatic blood clearance (Qh) of greater than 88% [1]
.
Murine Plasma Exposure (Satellite PK): Due to its high clearance, high doses were required to maintain exposure in efficacy studies. In Balb-C mice dosed intraperitoneally with 100 mg/kg of eut-22 (formulated in 30% cremophore), the plasma concentration was 7.6 ± 4.5 μM at 1 hour post-dose, but dropped to 0.2 ± 0.2 μM by 7 hours post-dose. For the 30 mg/kg dose, concentrations were 3.7 ± 0.4 μM at 1 hour and not detected by 7 hours. Similar exposure profiles were observed for the inactive enantiomer dis-22 [1]
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Plasma Protein Binding: Both eut-22 and dis-22 showed high plasma protein binding in mice, measured at 99% [1]
.

Structural Liabilities Removed: The initial hit compound 1 contained structural features with potential toxicity, mutagenicity, and drug-drug interaction liabilities: a nitro group (on the imidazole, potentially forming reactive metabolites) and an aniline (associated with hepatotoxicity, carcinogenicity). The optimization process that led to cut-22 successfully eliminated both of these structural alerts by replacing the nitroimidazole with a cyanopyrrole and the aniline sulfonamide with an indole sulfonamide [1]
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Plasma Protein Binding: eut-22 and dis-22 exhibit 99% plasma protein binding in mice [1]
.

[1]. N-Arylsulfonyl-α-amino carboxamides are potent and selective inhibitors of the chemokine receptor CCR10 that show efficacy in the murine DNFB model of contact hypersensitivity. Bioorg Med Chem Lett. 2016 Nov 1;26(21):5277-5283.

Background and Rationale: CCR10 is a chemokine receptor that plays an important role in the migration of skin-homing memory T-cells to the skin via its activation by the chemokine CCL27. Both CCR10 and CCL27 are associated with inflammatory skin diseases like allergic contact dermatitis and psoriasis. Interrupting the CCL27-CCR10 interaction has been proposed as a potential treatment for these conditions. This work describes the first potent and selective small molecule antagonists of CCR10 to test this hypothesis [1]
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Compound Designation and Stereochemistry: The optimized compound from the medicinal chemistry effort is referred to as cut-22 in the text. It is a racemic mixture. Its two enantiomers are eut-22 (the active, eutomer) and dis-22 (the inactive, distomer). The efficacy studies were performed with the active enantiomer, eut-22 [1]
.
Mechanism of Action: cut-22 acts as a potent and selective antagonist of the CCR10 chemokine receptor. It binds directly to CCR10, preventing the natural chemokines CCL27 and CCL28 from activating the receptor. This inhibition blocks downstream signaling events such as G-protein coupling, Ca²⁺ flux, and cAMP production, ultimately preventing CCR10-mediated cell chemotaxis [1]
.
Therapeutic Potential: The efficacy of eut-22 in the murine DNFB contact hypersensitivity model provides evidence that CCR10 antagonism is a valid therapeutic approach for treating dermatological inflammatory conditions like contact dermatitis and potentially psoriasis. The authors also suggest these inhibitors could be useful for studying the role of CCR10 in mucosal inflammation (e.g., asthma) and cancer [1]
.

C23H27N5O3S

453.557183504105

453.183

2040401-92-9

2040401-92-9; 1191030-85-9 (recamic);

131801164

White to off-white solid powder

2.6

2

5

7

32

811

1

S(C1=CC=CC2=C1C=CN2)(N[C@H](CCN1C=CC=C1C#N)C(N1CCC(C)CC1)=O)(=O)=O

BRJXJOWXAFLRTE-OAQYLSRUSA-N

InChI=1S/C23H27N5O3S/c1-17-8-13-28(14-9-17)23(29)21(10-15-27-12-3-4-18(27)16-24)26-32(30,31)22-6-2-5-20-19(22)7-11-25-20/h2-7,11-12,17,21,25-26H,8-10,13-15H2,1H3/t21-/m1/s1

N-[(2R)-4-(2-Cyanopyrrol-1-yl)-1-(4-methylpiperidin-1-yl)-1-oxobutan-2-yl]-1H-indole-4-sulfonamide

eut22BI6901 eut-22BI 6901 eut 22BI-6901

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 (e.g. under nitrogen), 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)

DMSO : ~160 mg/mL (~352.76 mM)

Solubility in Formulation 1: 5 mg/mL (11.02 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 4 mg/mL (8.82 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 40.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 4 mg/mL (8.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 40.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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