GPR35

Given that esterified carboxylic acid groups effectively improved cLogP and maintained agonistic activity, the carboxylic acid groups of the optimal compound 4 were esterified with different alkyl groups to yield compounds 13–17 (Table 3). Notablely, replacement of di-carboxyl with methyl esters (13) resulted in lower EC50 values in all three orthologs compared to 4b/Diethyl-Lodoxamide, but the Emax was declined to a certain extent. In order to further elevate the lipophilicity, several cyclic and larger alkyls were then attempted (compound 14–17). Unfortunately, these substituents proved to be unfavourable to the agonistic activity at least in one of the three orthologs. Among all the synthesized compounds, 4b showed the best properties, including high and equivalent potency in three orthologs of GPR35, high E max values and appropriate lipophilicity. Consequently, compound 4b was then selected for in vivo evaluation in DSS-induced mouse model of colitis to check its potential for the treatment of IBD [1].

In vivo efficacy of compound 4b/Diethyl-Lodoxamide in mouse model of DSS-induced colitis [1]
DSS treatment caused significant body weight loss, change in stool consistency, and rectal bleeding compared with the naive group, indicating active colitis (Fig. 2). 5-ASA, a currently marketed anti-IBD drug, was employed as a positive control. Compared with the vehicle group, treatment with 5-ASA (200 mg/kg) or compound 4b (20 mg/kg) attenuated the colitis symptoms of the diseased mice, including body weight loss, stool thinning, rectal bleeding and the overall DAI score (sum of scores for body weight loss, stool consistency, and rectal bleeding) (Fig. 2A-D).
Compound 4b treatment also markedly reduced colon shortening (Fig. 3A, B). On day 7, the colon length of the vehicle group was 5.1 ± 0.3 cm. Daily oral administration of 20 mg/kg compound 4b significantly improved the colon length (6.2 ± 0.3 cm). Histopathological analysis of the colon tissue revealed that compound 4b also reduced inflammatory cell infiltration, crypt loss and mucosal damage, resulting in a lower histological score (Fig. 3C, D).
Colitis can lead to increased production of pro-inflammatory cytokines or chemokines. Pro-inflammatory cytokines, such as IL-6, IL-1β, and chemokines, including CXCL1, CXCL2 and CCL2, were significantly reduced in colon tissues by compound 4b treatment (Fig. 4A, B). This demonstrates that compound 4b can reduce the degree of inflammation and infiltration of inflammatory cells in colon tissue.
These results establish that GPR35 agonist compound 4b can ameliorate clinical symptoms and colon tissue damage in mice with colitis, providing a protective role in intestinal inflammation.

Calcium mobilization assay [1]
The human, mouse or rat GPR35 gene was sub-cloned in vector pcDNA3.0, and sequences were verified by DNA sequencing. HEK293 cells were cultured in 1x DMEM supplemented with 10 % (v/v) fetal bovine serum, and incubated in 5 % CO2 at 37 °C. In subsequent transient transfection, approximately 2 × 106 cells were mixed with 2 μg plasmids in 200 μL transfection buffer. Electroporation was performed with a Scientz-2C electroporation apparatus.
HEK293 cells expressing GPR35 and Gα16 were seeded in 96-well plates at a density of 3 × 104 cells/well, and cultured overnight. The cells were then incubated with 2 μmol/L Fluo-4 AM in HBSS (5.4 mmol/L KCl, 0.3 mmol/L Na2HPO4, 0.4 mmol/L KH2PO4, 4.2 mmol/L NaHCO3, 1.3 mmol/L CaCl2, 0.5 mmol/L MgCl2, 0.6 mmol/L MgSO4, 137 mmol/L NaCl, 5.6 mmol/L d-glucose, and 250 μmol/L sulfinpyrazone, pH 7.4) at 37 °C for 45 min. After thorough washing, 50 μL HBSS was added. Following incubation at room temperature for 10 min, 25 μL agonist was dispensed into the well using a FlexStation III microplate reader, and the intracellular calcium change was recorded at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The EC50 and Emax values for each curve were calculated by Prism 5.0 software.

A mouse model of dextran sulphate sodium salt (DSS)-induced colitis [1]
The DSS-induced colitis model in mice was established as reported previously22, 23. In this study, mice were treated with 2.5 % (w/v) DSS in drinking water for 5 days, followed by DSS-free water for an additional 2 days. The severity of colitis was scored daily by recording standard parameters, including body weight, diarrhea, and bloody stools. The disease activity index (DAI) was measured following the procedure reported elsewhere.24 With regard to drug treatment, mice were treated once daily by oral gavage with vehicle (CMCNa, 0.5 % w/v), compound 4b/Diethyl-Lodoxamide (20 mg/kg) or 5-ASA (positive control, 200 mg/kg) for 7 days, starting at day 1 of the DSS treatment. Control mice were administered water as a negative control. On day 7, the entire colon was removed from all the sacrificed mice, and the length of the colon measured and recorded. Each colon sample was divided into two parts. One part of the colon samples was used for histological analysis, and the second part was used for a quantitative real-time (qRT)-PCR assay.

We also attempted to evaluate the pharmacokinetic characteristics of compound 4b/diethyllodoxamide. However, after oral administration of 20 mg/kg of compound 4b to mice, the compound could not be detected in mouse plasma samples. Subsequent in vitro tests using isolated mouse plasma revealed that compound 4b was very unstable and disappeared within minutes of incubation with plasma. These results suggest that compound 4b may exert its effects in local intestinal tissues before being absorbed into the bloodstream. [1]

[1]. Discovery of a novel GPR35 agonist with high and equipotent species potency for oral treatment of IBD. Bioorg Med Chem. 2023 Dec 15:96:117511.

protein-coupled receptor 35 (GPR35) has been identified as a potential therapeutic target for inflammatory bowel disease (IBD). However, the lack of agonists with potent and equivalent activity against GPR35 in both humans and mice has limited in vivo studies of GPR35 agonists in mouse models of IBD. This study obtained a series of agonists with potent and equivalent activity against GPR35 in humans, mice, and rats through structural modification of loduxamide. These molecules eliminated the species selectivity of human, mouse, and rat homologs commonly found in GPR35 agonists, including loduxamide. Furthermore, the cLogP properties were optimized to better conform to drug-likeness rules, ultimately yielding diethyl-loduxamide/compound 4b (cLogP = 2.41), with EC50 values of 76.0 nM, 63.7 nM, and 77.8 nM against GPR35 in humans, mice, and rats, respectively. Oral administration of compound 4b (20 mg/kg) alleviated clinical symptoms of DSS-induced IBD in mice, with slightly better efficacy than 5-ASA (200 mg/kg). In summary, this compound can serve as a new starting point for developing more effective and species-independent GPR35 agonists for the treatment of IBD and deserves further investigation. [1] Since the discovery of GPR35, a variety of highly effective agonist structures have been identified and synthesized, but none of these molecules can equivalently activate GPR35 in humans, mice, and rats. We modified the structure of lodoxam to obtain a series of molecules with high in vitro activity and equivalent efficacy, which can be used as tool compounds. By replacing the cyano group of lodoxam with trifluoromethyl, we obtained the best compound 4. This is the first molecule with EC50 values of less than 100 nM for human, mouse, and rat GPR35. However, the cLogP value of compound 4 is -1.01. We further substituted the dicarboxyl group of this molecule with a lipophilic ester group to obtain diethyllodoxaamide compounds 4b and 13-17 with optimized lipophilicity. These molecules exhibited cLogP values in the range of 2-5 while maintaining high and equivalent activity against human and mouse GPR35. The optimal compound 4b showed EC50 values of 76.0 nM, 63.7 nM, and 77.8 nM against human, mouse, and rat GPR35, respectively; Emax values were in the range of 80-105%, and cLogP = 2.41. We selected diethyllodoxaamide/compound 4b and investigated its efficacy in IBD mice via oral administration. Previous studies have shown that intraperitoneal injection of 100 mg/kg pamoate effectively alleviated DSS-induced colitis symptoms in mice. However, there are currently no reports in the literature regarding the effects of oral GPR35 agonists on IBD mice. We are the first to report that oral administration of the GPR35 agonist compound 4b/diethyllodoxaamide can reduce the severity of DSS-induced IBD in mice. A dose of 20 mg/kg of compound 4b not only significantly reduced the Disease Activity Index (DAI) score but also increased colon length and reduced tissue damage and inflammation, with efficacy slightly superior to 200 mg/kg of 5-aminosalicylic acid (5-ASA). Compound 4b represents a novel class of neutral ester GPR35 agonists, distinct from acidic lodoxaamide, and thus provides a new starting point for the discovery and development of more effective, species-neutral GPR35 agonists for the treatment of IBD. However, given the instability of 4b in plasma, much structural modification work is still needed in the future. [1]

C15H14CLN3O6

367.74

53882-13-6

40915

Typically exists as solids at room temperature

1.43g/cm3

1.571

1.361

2

7

8

25

545

0

CCOC(=O)C(=O)NC1=CC(=CC(=C1Cl)NC(=O)C(=O)OCC)C#N

LODOXAMIDE ETHYL; 53882-13-6; Lodoxamide Diethyl Ester; Lodoxamide ethyl [USAN]; U-42718; U-42,718; ethyl 2-[2-chloro-5-cyano-3-[(2-ethoxy-2-oxoacetyl)amino]anilino]-2-oxoacetate; TQY1B8145B;

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.)