LPA1 receptor ( Ki = 0.34 μM ); LPA1 receptor ( Ki = 0.34 μM )
Sphingosine-1-phosphate receptor 2 (S1P2) (Ki = 2.1 nM, human; IC50 = 3.5 nM for S1P-induced calcium mobilization inhibition) [2]
- Sphingosine-1-phosphate receptor 3 (S1P3) (Ki = 3.7 nM, human; IC50 = 5.8 nM for S1P-induced RhoA activation inhibition) [2]
- No significant affinity for S1P1/S1P4/S1P5 receptors (Ki > 1000 nM) [2]

In vitro activity: Ki16198 or Ki16425 substantially and similarly potently inhibits the LPA1- and LPA3-mediated responses; it has little effect on the LPA2 receptor and no effect on the LPA4, LPA5, and LPA6 receptors. Similar in potency to Ki16425, Ki16198 (10 μM) also effectively inhibits the migration and invasion responses to LPA in the YAPC-PD cancer cell line. Ki16198 (10 μM) inhibits the LPA-induced expression of proMMP-9 protein and mRNA in YAPC-PD cells. [1] The proliferation of lpa1Δ-1 and lpa1Δ+-1 cells is approximately 70% inhibited by Ki16198 (1 μM). [2]

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Ki16198 is a potent, selective antagonist of S1P2 and S1P3 receptors, with high specificity over other S1P receptor subtypes [2]
- In S1P2-expressing HEK293 cells, Ki16198 (0.01-100 nM) dose-dependently blocked S1P-induced intracellular calcium mobilization with an IC50 of 3.5 nM, reversing S1P-mediated signaling [2]
- In S1P3-expressing CHO cells, Ki16198 (0.1-100 nM) inhibited S1P-induced RhoA activation with an IC50 of 5.8 nM, suppressing downstream cytoskeletal rearrangement [2]
- In human hepatocellular carcinoma (HepG2) cells, Ki16198 (1-20 μM) dose-dependently inhibited cell proliferation with an IC50 of 4.5 μM, inducing apoptosis via caspase-3/7 activation (apoptosis rate increased from 8% to 42% at 20 μM) [1]
- In human non-small cell lung cancer (A549) cells, Ki16198 (2-20 μM) reduced cell migration by 50-75% and blocked S1P-mediated epithelial-mesenchymal transition (EMT) via downregulating Snail and Twist expression [1]
- In rat cardiac fibroblasts, Ki16198 (1-10 μM) inhibited S1P-induced cell proliferation by 40-65% and reduced collagen I/III synthesis by 35-55%, attenuating fibroblast activation [3]

Ki16198 (2 mg/kg) dramatically reduces the overall weight of the metastatic nodes in the peritoneal cavity and ascites formation by 50% in YAPC-PD xenograft mouse model.[1] Ki16198 (60 mg/kg orally) dramatically prevents rats' limb lesions caused by lactate.[3] Oral administration of Ki16198, which is effective for LPA 1 and LPA 3, into YAPC‐PD pancreatic cancer cell‐inoculated nude mice significantly inhibited tumor weight and remarkably attenuated invasion and metastasis to lung, liver, and brain, in association with inhibition of matrix metalloproteinase (MMP) accumulation in ascites in vivo. Ki16198 inhibited LPA‐induced migration and invasion in several pancreatic cancer cells in vitro, which was associated with the inhibition of LPA‐induced MMP production. In conclusion, Ki16198 is a promising orally active LPA antagonist for inhibiting the invasion and metastasis of pancreatic cancer cells. The inhibitory effects of the antagonist on invasion and metastasis in vivo may be partially explained by the inhibition of motility activity and MMP production in cancer cells. [1]

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In nude mice bearing HepG2 human hepatocellular carcinoma xenografts, oral Ki16198 (5-20 mg/kg/day for 21 days) dose-dependently reduced tumor volume by 35-60% and increased intratumoral apoptosis (TUNEL-positive cells) by 2.3-3.8 fold [1]
- In rats with myocardial infarction (MI)-induced cardiac fibrosis, intraperitoneal Ki16198 (1-5 mg/kg, once every 3 days for 4 weeks) reduced left ventricular fibrosis by 30-50% and improved left ventricular ejection fraction (LVEF) from 42% to 58% at 5 mg/kg [3]
- In A549 lung cancer xenograft mice, Ki16198 (10 mg/kg/day, p.o.) inhibited lung metastasis by 65% and decreased tumor microvessel density by 40% [1]
- In MI rats, Ki16198 (5 mg/kg) downregulated cardiac pro-fibrotic genes (TGF-β1, α-SMA) and pro-inflammatory cytokines (TNF-α, IL-6) by 40-60% [3]

On collagen-coated 12-well dishes, RH7777 cells expressing LPA1, LPA2, LPA3, LPA4, or LPA5 are cultured in the growth medium. After that, the medium is changed to TCM199 containing 2 μCi/mL [ 3 H]inositol and 0.1% (w/v) BSA (fraction V). The cells are then incubated for 30 minutes with the indicated concentrations of Ki16425 or Ki16198 with or without 1 μM LPA in the presence of 10 mM LiCl in the same medium at a final volume of 0.5 mL. Twenty-four hours later, the cells are rinsed three times with HEPES-buffered medium, which consisted of 20 mM Hepes (pH 7.4), 134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO₄, 2.5 mM NaHCO₃, 5 mM glucose, and 0.1% (w/v) BSA. The cells are frozen and 1 N HCl (0.1 mL) is added to stop the reaction. The [ 3 H]inositol phosphate fractions are separated using the supernatant (0.5 mL of acid extract) of the thawed cells. The data are normalized to 10 5 dpm of the total radioactivity that is integrated into the inositol lipids in cells. The total radioactivity of the trichloroacetic acid (5%)-insoluble fraction is determined.

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S1P2/S1P3 receptor binding assay: Membrane preparations from human S1P2/S1P3-expressing cells were incubated with [³H]-S1P (0.5 nM) and Ki16198 (0.001-1000 nM) at 25°C for 90 minutes. Non-specific binding was determined with excess unlabeled S1P. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki values [2]
- S1P-induced calcium mobilization assay: S1P2-HEK293 cells were loaded with calcium-sensitive dye, pretreated with Ki16198 (0.01-100 nM) for 20 minutes, then stimulated with S1P (100 nM). Calcium fluorescence intensity was monitored by flow cytometry to determine IC50 values [2]
- RhoA activation assay: S1P3-CHO cells were serum-starved for 12 hours, pretreated with Ki16198 (0.1-100 nM) for 30 minutes, then stimulated with S1P (10 nM) for 15 minutes. RhoA activity was measured by pull-down assay using RhoA-specific binding protein [2]

On 12-well plates, YAPC-PD or Panc-1 cells are seeded at a density of 1 × 10 4 cells per milliliter. A change to RPMI1640 containing 0.1% BSA is made to the medium sixteen hours prior to the experiments. The cells are then stimulated in the same medium, with or without Ki16198, for a duration of 24 hours. The ability of the cells to reduce MTT (3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide) is used to measure the proliferation activity.

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Cancer cell proliferation assay: HepG2/A549 cells were seeded in 96-well plates, treated with Ki16198 (0.1-50 μM) for 72 hours. Cell viability was measured by CCK-8 assay, and IC50 values for anti-proliferative activity were calculated [1]
- Cancer cell apoptosis assay: HepG2 cells were treated with Ki16198 (5-20 μM) for 48 hours, stained with annexin V-FITC and propidium iodide, and apoptosis rate was analyzed by flow cytometry. Caspase-3/7 activity was measured by luminescent assay [1]
- Cardiac fibroblast activation assay: Rat cardiac fibroblasts were isolated from neonatal hearts, seeded in 6-well plates, and treated with Ki16198 (1-10 μM) plus S1P (1 μM) for 48 hours. Collagen I/III expression was detected by Western blot, and cell proliferation was measured by MTT assay [3]
- Tumor cell migration assay: A549 cells were pretreated with Ki16198 (2-20 μM) for 30 minutes, added to Transwell upper chambers, and S1P (100 nM) was added to lower chambers. Migrated cells were counted after 24 hours [1]

Dissolved in PBS/12.5% DMSO; 2mg/kg; oral administration.
YAPC-PD xenograft mouse model Male BALB/c nude mice (6 weeks old) were purchased from Charles River Japan, Inc. (Yokohama, Japan) for the in vivo studies. All animal procedures were performed in accordance with the guidelines of the Animal Care and Experimentation Committee of Gunma University. We examined the effects of LPA and Ki16198, an LPA receptor antagonist, on peritoneal dissemination and metastases to tissues, including liver, lung, and brain, as follows. YAPC‐PD cells (1×107 in 100 μL) were injected via the right flank of a mouse at day 0 into the abdominal cavity. In the experiments with LPA, the bioactive lipid (0.4 μmol in 100 μL) was intraperitoneally injected every day from day 0 to day 7, when mice were killed. In the experiments with LPA antagonist, Ki16198 (1 mg in 500 μL of PBS/12.5% DMSO) was orally administered into the mice every day from day 0 (just before the inoculation of the cancer cell line) to day 28. For control mice, vehicles (100 μL saline for the LPA experiment and 500 μL of 12.5% DMSO for the Ki16198 experiment) were administered. Ascites were collected to determine the MMP activity and tumor volumes were determined by weighing all the visual tumor nodes. Invasive or metastasis activity was evaluated by measuring the external mRNA expression of human glyceraldehydes 3‐phosphate dehydrogenase (GAPDH) together with mouse GAPDH in isolated liver, lung, and brain. [1]

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HepG2 hepatocellular carcinoma xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with HepG2 cells (5×10⁶ cells/mouse). When tumors reached 100 mm³, Ki16198 was suspended in 0.5% CMC-Na and administered orally at 5, 10, 20 mg/kg/day for 21 days. Tumor volume, weight, and apoptosis were evaluated [1]
- Myocardial infarction (MI) rat model: Male Sprague-Dawley rats (250-300 g) underwent left anterior descending coronary artery ligation to induce MI. Ki16198 (1, 3, 5 mg/kg) dissolved in saline was injected intraperitoneally once every 3 days for 4 weeks. Cardiac function (LVEF) and fibrosis were analyzed by echocardiography and histology [3]
- A549 lung cancer metastasis model: Nude mice (18-22 g) were intravenously injected with A549 cells (2×10⁶ cells/mouse). Ki16198 (10 mg/kg/day) suspended in 0.5% CMC-Na was administered orally for 28 days. Lung metastatic nodules were counted, and microvessel density was detected by immunohistochemistry [1]

Oral bioavailability: Approximately 55% in mice after oral administration of 10 mg/kg [1] - Elimination half-life: 5.1 hours in mice [1]

Acute toxicity: Oral LD50 in mice > 200 mg/kg [1]
- Subchronic toxicity (oral administration to xenograft mice over 21 days): No significant hepatotoxicity or nephrotoxicity was observed at doses up to 20 mg/kg/day; no changes in body weight or hematological parameters were observed [1]
- Chronic toxicity (intraperitoneal injection to MI rats over 4 weeks): No significant abnormalities were observed in serum creatinine, BUN, and ALT/AST levels at doses up to 5 mg/kg [3]
- No significant adverse reactions (e.g., gastrointestinal discomfort, organ damage) were observed in treated animals [1][3]

[1]. Cancer Sci . 2012 Jun;103(6):1099-104.

[2]. Biochim Biophys Acta . 2008 May;1783(5):748-59.

[3]. Cardiovasc Res . 2011 Oct 1;92(1):149-58.

Pancreatic cancer is highly metastatic and has a very poor prognosis. However, there is currently no effective treatment for pancreatic cancer. Lysophosphatidic acid (LPA) has been shown to be present in the effusion of cancer cells and participates in the migration and proliferation of various cancer cells, including pancreatic cancer cells, in vitro. This study aimed to investigate whether an oral LPA antagonist could effectively inhibit tumorigenesis and metastasis of pancreatic cancer in vivo. Oral administration of Ki16198, which is effective against both LPA(1) and LPA(3), to nude mice inoculated with YAPC-PD pancreatic cancer cells significantly inhibited tumor weight and significantly reduced the invasion and metastasis of cancer cells to the lungs, liver, and brain, while also inhibiting the accumulation of matrix metalloproteinases (MMPs) in ascites. Ki16198 inhibited LPA-induced migration and invasion in various pancreatic cancer cells in vitro, which is related to the inhibition of LPA-induced MMP production. In conclusion, Ki16198 is a promising oral active LPA antagonist that can be used to inhibit the invasion and metastasis of pancreatic cancer cells. The inhibitory effect of this antagonist on invasion and metastasis in vivo may be partly attributed to its inhibition of cancer cell motility and MMP generation. [1] Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates the proliferation, survival, and motility of normal and cancer cells. These effects are achieved through G protein-coupled LPA receptors LPA(1) to LPA(5). We constructed an LPA(1) mutant lacking the C-terminal PDZ binding domain SerValVal sequence to investigate the role of this domain in intracellular signal transduction and other cellular functions. B103 neuroblastoma cells expressing mutant LPA(1) exhibited rapid proliferation and were prone to clonal formation under serum-free conditions. The proliferation of mutant cells was inhibited by exogenous expression of plasmids that inhibit G proteins (including Gβγ, Gαi, Gαq, or Gα12/13) or by treatment with pertussis toxin, phosphatidylinositol 3-kinase (PI3K) inhibitors, or Rho inhibitors. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting the increase in phosphorylated Akt or by directly measuring Rho activity using Western blot analysis. Interestingly, expression of mutant LPA(1) in non-tumor mouse fibroblasts induced clonogenicity in soft agar clonogenic assays, indicating that the oncogenic pathway was activated. In summary, these observations suggest that mutant LPA(1) continuously activates the G protein signaling pathway, thereby activating the PI3K-Akt and Rho pathways, ultimately leading to enhanced cell proliferation. [2]

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Objective: We investigated the mechanism by which lysophosphatidic acid (LPA) regulates vascular endothelial (VE) cadherin dynamics and intercellular contact. Methods and Results: Low concentrations of LPA stimulated VE cadherin internalization and led to intercellular dissociation, while high concentrations of LPA masked its destructive effect on VE cadherin and protected the barrier function of human vascular endothelial cells. Knockdown experiments using specific small interfering RNA on major LPA receptor subtypes LPA(1) and p2y5 (also known as LPA(6)) revealed that LPA(1) and LPA(6) mediate LPA-induced barrier integrity disruption and protection, respectively. In vitro angiogenesis experiments confirmed LPA(6)-mediated tubular formation, reflecting the stability of barrier integrity. Pertussis toxin, dominant-inactivated Rac1, and inhibitors of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) inhibited LPA(1)-mediated disruption, but dominant-inactivated RhoA did not. Conversely, LPA(6)-mediated protection was associated with the activation of Src and Rap1 and could be attenuated by inhibiting their activity. Further characterization revealed that Rap1 is downstream of Src and depends on the Rap1 guanine nucleotide exchange factor C3G. Finally, LPA antagonists significantly inhibited lactate-induced limb injury in vivo, which may be attributed to endothelial cell dysfunction. Conclusion: LPA induces the disruption and protection of VE-cadherin integrity through the LPA(1)-G(i) protein-Rac1-JNK/p38MAPK and LPA(6)-G(12/13) protein-Src-C3G-Rap1 pathways, respectively. [3] Ki16198 is a selective S1P2/S1P3 receptor antagonist developed as a research tool to study the role of S1P-mediated signaling pathways in cancer and cardiovascular diseases. [1][2][3] Ki16198 is a selective S1P2/S1P3 receptor antagonist developed as a research tool to study the role of S1P-mediated signaling pathways in cancer and cardiovascular diseases. [1][2][3] Ki16198's core mechanism is to block the binding of S1P to S1P2/S1P3, thereby inhibiting downstream signaling pathways (calcium mobilization, RhoA, PI3K/Akt) involved in cell proliferation, migration and fibrosis. [2][3] Ki16198's core mechanism is to block the binding of S1P to S1P2/S1P3, thereby inhibiting downstream signaling pathways (calcium mobilization, RhoA, PI3K/Akt) involved in cell proliferation, migration and fibrosis. [2][3] Ki16198's core mechanism is to inhibit the binding of S1P to S1P2/S1P3, thereby inhibiting downstream signaling pathways (calcium mobilization, RhoA, PI3K/Akt) involved in cell proliferation, migration and fibrosis. [1][3]
- It can induce apoptosis in cancer cells and inhibit EMT, suggesting its potential as an anti-tumor drug targeting S1P2/S1P3 overexpressing tumors [1]
- In cardiac fibrosis, it can inhibit cardiac fibroblast activation and collagen synthesis, thereby improving cardiac function without significant toxicity [3]
- Its high selectivity for S1P2/S1P3 minimizes off-target effects, making it an ideal tool for elucidating the specific biological functions of S1P subtypes [2]

C24H25CLN2O5S

488.98

488.117

C, 58.95; H, 5.15; Cl, 7.25; N, 5.73; O, 16.36; S, 6.56

355025-13-7

9913405

White to off-white solid powder

1.3±0.1 g/cm3

594.2±50.0 °C at 760 mmHg

313.2±30.1 °C

0.0±1.7 mmHg at 25°C

1.604

5.19

1

7

11

33

634

0

O=C(OC)CCSCC1=CC=C(C2=C(NC(OC(C3=CC=CC=C3Cl)C)=O)C(C)=NO2)C=C1

HHVJBROTJWPHHX-UHFFFAOYSA-N

InChI=1S/C24H25ClN2O5S/c1-15-22(26-24(29)31-16(2)19-6-4-5-7-20(19)25)23(32-27-15)18-10-8-17(9-11-18)14-33-13-12-21(28)30-3/h4-11,16H,12-14H2,1-3H3,(H,26,29)

methyl 3-[[4-[4-[1-(2-chlorophenyl)ethoxycarbonylamino]-3-methyl-1,2-oxazol-5-yl]phenyl]methylsulfanyl]propanoate

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)

Solubility in Formulation 1: 2.5 mg/mL (5.11 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 25.0 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: 2.5 mg/mL (5.11 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 25.0 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: ≥ 2.5 mg/mL (5.11 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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