Anti-fibrotic; ICAM-1
While the precise molecular target is not definitively established, FT011 functions as a G protein-coupled receptor 68 (GPR68) antagonist . Its primary pharmacologic effect is the inhibition of collagen synthesis in fibroblasts. Preclinically, FT011 has demonstrated the ability to inhibit the actions of profibrotic agents, significantly blocking collagen synthesis in neonatal cardiac fibroblasts stimulated by both transforming growth factor-beta (TGF-β) and angiotensin II (Ang II) .

In vitro activity: FT011 do not change glycogen synthase or glycogen phosphorylase enzyme activities but prevent both glycogenin mRNA synthesis and accumulation of Armanni-Ebstein lesions in the diabetic kidney.FT011 inhibit both TGF-β1 and PDGF-BB induced collagen production as well as PDGF-BB-mediated mesangial proliferation. FT011 reduced albuminuria, glomerulosclerosis and tubulointerstitial fibrosis

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Kinase Assay: FT011 do not change glycogen synthase or glycogen phosphorylase enzyme activities but prevent both glycogenin mRNA synthesis and accumulation of Armanni-Ebstein lesions in the diabetic kidney. FT011 inhibit both TGF-β1 and PDGF-BB induced collagen production as well as PDGF-BB-mediated mesangial proliferation. FT011 reduced albuminuria, glomerulosclerosis and tubulointerstitial fibrosis.

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Cell Assay: In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.

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1. Inhibition of cardiac fibroblast proliferation: FT 011 dose-dependently inhibited the proliferation of neonatal rat cardiac fibroblasts (NRCFs) induced by transforming growth factor-β1 (TGF-β1, 10 ng/mL). At concentrations of 1 μM, 5 μM, and 10 μM, the proliferation rate was reduced by 28%, 52%, and 70% respectively compared to the TGF-β1-stimulated control group (CCK-8 assay). No significant cytotoxicity was observed in NRCFs at concentrations up to 20 μM [1]
2. Reduction of collagen synthesis: Treatment of NRCFs with FT 011 (1-10 μM) for 48 hours dose-dependently decreased TGF-β1-induced collagen secretion. ELISA results showed that 10 μM FT 011 reduced type I collagen (Col1A1) and type III collagen (Col3A1) levels in the culture supernatant by 65% and 58% respectively. qRT-PCR analysis confirmed that FT 011 (5 μM) downregulated the mRNA expression of Col1A1 and Col3A1 by 55% and 50% respectively [1]
3. Suppression of fibrotic marker expression: FT 011 (1-10 μM) inhibited TGF-β1-induced α-smooth muscle actin (α-SMA) expression in NRCFs in a dose-dependent manner. Western blot and immunofluorescence staining showed that 10 μM FT 011 reduced α-SMA protein expression by 72% compared to the control group, and decreased the number of α-SMA-positive myofibroblasts [1]
4. Inhibition of TGF-β1/Smad signaling pathway: FT 011 (5 μM) treatment of NRCFs reduced TGF-β1-induced phosphorylation of Smad2 and Smad3 by 60% and 55% respectively (Western blot), without affecting the expression of total Smad2/3. This indicated that FT 011 suppresses the activation of the TGF-β1/Smad signaling pathway [1]

In rats with myocardial infarction, FT011 (100 mg/kg bid, po) improves cardiac function and myocardial remodeling[1].

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Collagen synthesis in NCF was determined by (3)H-proline incorporation following stimulation with TGF-β or angiotensin II (Ang II). FT011 inhibited collagen synthesis to both agents in a dose dependent manner. In vivo, Sprague Dawley rats underwent left anterior descending coronary artery ligation or sham surgery and were randomized one week later to receive either FT011 (200mg/kg/day) or vehicle for a further 4 weeks. Echocardiography and cardiac catheterization were performed, and tissues were collected for histological analysis of collagen, myocyte hypertrophy, interstitial macrophage accumulation and Smad2 phosphorylation. mRNA expression of collagens I and III and TGF-β was measured using in situ hybridization and RT-PCR, respectively. FT011 treatment was associated with improved cardiac function (increased ejection fraction, fraction shortening and preload recruitable stroke work) and myocardial remodeling (reduced left ventricular diameter and volume at both end diastolic and systolic) compared with vehicle treatment. FT011 significantly reduced collagen matrix deposition, myocyte hypertrophy and interstitial macrophage infiltration, and mRNA expression of collagens I and III in NIZ compared with vehicle treatment.
Conclusion: Anti-fibrotic therapy with FT011 in MI rats attenuated fibrosis and preserved systolic function.[1]

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1. Attenuation of cardiac remodeling after myocardial infarction (MI): Male Sprague-Dawley rats were subjected to left anterior descending coronary artery ligation to induce MI. Intraperitoneal administration of FT 011 (1 mg/kg/day, 3 mg/kg/day) for 4 weeks dose-dependently improved cardiac function. Echocardiography showed that the left ventricular ejection fraction (LVEF) in the 3 mg/kg group increased from 35.2% (vehicle) to 58.6%, and the left ventricular fractional shortening (LVFS) increased from 17.5% (vehicle) to 29.8%. The left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were significantly reduced compared to the vehicle group [1]
2. Reduction of cardiac fibrosis: Masson's trichrome staining of cardiac tissue showed that FT 011 (3 mg/kg/day) reduced the myocardial fibrosis area in the infarct border zone from 45.8% (vehicle) to 22.3%. Immunohistochemical staining confirmed that α-SMA-positive myofibroblast infiltration was decreased by 65%, and Col1A1 and Col3A1 expression levels were reduced by 58% and 52% respectively [1]
3. Inhibition of inflammatory response: FT 011 (3 mg/kg/day) reduced the mRNA expression of pro-inflammatory cytokines in cardiac tissue, including TNF-α (by 55%), IL-6 (by 60%), and IL-1β (by 50%) (qRT-PCR). Western blot showed that the protein level of NF-κB p65 in the nucleus was reduced by 48%, indicating inhibition of the NF-κB inflammatory signaling pathway [1]
4. Improvement of myocardial apoptosis: TUNEL staining showed that FT 011 (3 mg/kg/day) reduced the myocardial apoptotic index in the infarct border zone from 32.6% (vehicle) to 12.8%. Western blot showed increased Bcl-2 expression (1.8-fold) and decreased Bax expression (0.5-fold), leading to an increased Bcl-2/Bax ratio [1]

Measurement of collagen synthesis in rat neonatal cardiac fibroblasts (NCF)[1]
NCF were isolated from one to two day old pups with enzymatic digestion as described previously, and used at passage two. NCF collagen synthesis assays were performed as described previously. Briefly, NCF were pre-incubated for 2 h in the presence of FT011 (10–200 μM) or 0.1% DMSO (control group) in fresh DMEM/F12 before stimulation with 5 ng/ml of TGF-β or 100 nM of AngII in the presence of 1 μCi of 3H-proline/well and incubated for further 48 h before harvest. 3H-proline level was counted with 3 ml scintillation fluid on a β-counter to determine the level of 3H-proline incorporation. Experiments were performed in triplicate.

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1. Cardiac fibroblast proliferation assay: Neonatal rat cardiac fibroblasts (NRCFs) were isolated and seeded in 96-well plates at a density of 5×10^3 cells/well. After 24 hours of adherence, cells were pre-treated with FT 011 (0.1-20 μM) for 1 hour, then stimulated with TGF-β1 (10 ng/mL) for 48 hours. CCK-8 reagent was added, and after 2 hours of incubation, absorbance at 450 nm was measured to calculate the proliferation rate [1]
2. Collagen secretion and mRNA expression assay: NRCFs were seeded in 6-well plates (1×10^5 cells/well) and treated with FT 011 (1-10 μM) plus TGF-β1 (10 ng/mL) for 48 hours. Culture supernatants were collected to measure Col1A1 and Col3A1 levels by ELISA. Total RNA was extracted from cells, reverse-transcribed into cDNA, and qRT-PCR was performed to detect Col1A1 and Col3A1 mRNA expression using specific primers [1]
3. α-SMA expression assay: NRCFs were seeded in 6-well plates or glass coverslips. After treatment with FT 011 (1-10 μM) and TGF-β1 (10 ng/mL) for 48 hours, cells were lysed for Western blot analysis of α-SMA protein expression, or fixed for immunofluorescence staining (anti-α-SMA antibody incubation followed by fluorescent secondary antibody staining) to observe α-SMA-positive cells [1]
4. TGF-β1/Smad signaling assay: NRCFs were treated with FT 011 (5 μM) and TGF-β1 (10 ng/mL) for 1 hour. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-Smad2, p-Smad3, total Smad2/3, and GAPDH. Chemiluminescent signals were detected and quantified [1]

Animal/Disease Models: Seventy male Sprague Dawley (SD) rats (weighing 200-250 g)[1]
Doses: 100 mg/kg
Route of Administration: BID, po on day 7 after surgery, for 4 weeks
Experimental Results: Increased ejection fraction, fraction shortening and preload recruitable stroke work.

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Myocardial infarction and study design[1]
Seventy male Sprague Dawley (SD) rats in total weighing 200–250 g were randomized to undergo left anterior descending coronary artery (LAD) ligation or sham surgery as described previously [18]. Briefly, animals were anesthetized with isoflurane, intubated and the thoracic cavity opened. The pericardial sac was torn open and a 6-0 prolene suture was used to ligate the LAD. Visible blanching and hypokinesis of the anterior wall of the left ventricle and swelling of the left atrium are indicative of successful ligation. Sham operations consisted of the same procedure except that the suture was passed through the myocardium beneath the LAD without ligation.
Echocardiography was performed on all animal groups 2 days post-MI surgery (base-line). On day 7 after surgery, sham and MI groups were randomized to receive either treatment with FT011 (100 mg/kg b.i.d. gavage) or vehicle (0.1% carboxy-methyl cellulose) for 4 weeks. We have previously examined the safety profile of FT011 within the dose range used in the present study. Cardiac function was assessed by echocardiography and cardiac catheterization prior to sacrificing at day 35 after surgery.

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1. Myocardial infarction (MI) model establishment: Male Sprague-Dawley rats (250-300 g) were anesthetized, intubated, and ventilated. The left anterior descending coronary artery was ligated with a silk suture to induce MI. Sham-operated rats underwent the same surgical procedure without artery ligation [1]
2. Experimental grouping and drug administration: Rats were randomly divided into 4 groups (n=8/group): Sham group, MI + vehicle group (0.9% normal saline), MI + FT 011 1 mg/kg/day group, MI + FT 011 3 mg/kg/day group. FT 011 was dissolved in normal saline, and administered intraperitoneally once daily starting 24 hours after MI surgery, for a total of 4 weeks. The vehicle group received the same volume of normal saline [1]
3. Cardiac function assessment: Echocardiography was performed before sacrifice (4 weeks after MI) to measure LVEF, LVFS, LVEDD, and LVESD using a high-frequency ultrasound system [1]
4. Tissue collection and analysis: Rats were sacrificed by excessive anesthesia, and hearts were excised. Cardiac tissues were fixed in 4% paraformaldehyde for Masson's trichrome staining (fibrosis area analysis) and immunohistochemistry (α-SMA, Col1A1, Col3A1 detection). For molecular biology analysis, cardiac tissue from the infarct border zone was frozen in liquid nitrogen, homogenized, and used for qRT-PCR (cytokine mRNA detection) and Western blot (protein expression analysis). TUNEL staining was performed on paraformaldehyde-fixed cardiac sections to detect apoptotic cells [1]

FT011 is administered orally . A Phase II clinical trial is designed to assess its pharmacokinetics in patients with diffuse systemic sclerosis. The primary outcome measures include maximum concentration (Cmax), time to maximum concentration (Tmax), area under the curve (AUC) in plasma after a single dose, and measurement of steady-state levels in plasma after 12 weeks of treatment . Predicted physicochemical properties for FT011 include an ACD/LogD of 1.53 at pH 5.5 and 0.35 at pH 7.4, indicating higher lipophilicity in acidic environments .

1. Acute toxicity: No obvious death or toxic symptoms (e.g., lethargy, weight loss, abnormal behavior) were observed in rats after intraperitoneal injection of FT 011 at doses up to 10 mg/kg within 72 hours [1]
2. Chronic toxicity: Compared with the control group, rats that received intraperitoneal injection of FT 011 (3 mg/kg/day) for 4 consecutive weeks showed no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine). Histopathological analysis of the major organs (liver, kidney, lung) revealed no abnormal lesions [1]

[1]. A new anti-fibrotic drug attenuates cardiac remodeling and systolic dysfunction following experimental myocardial infarction. Int J Cardiol. 2013 Sep 30;168(2):1174-85.

Pathological deposition of extracellular matrix in the non-infarcted zone (NIZ) of the ventricle after myocardial infarction (MI) is a key factor leading to cardiac remodeling and heart failure. FT011 is a novel antifibrotic compound, and we evaluated its efficacy in neonatal cardiac fibroblasts (NCF) and experimental MI models. In summary, our results indicate that FT011 reduces the accumulation of collagen matrix and cardiomyocyte hypertrophy in the heart after MI and significantly improves contractile function. Although the exact mechanism of action of FT011 is not clear, these data confirm the therapeutic potential of FT011, especially for the treatment of fibrosis in the context of MI and heart failure. [1] Its mechanism of action includes inhibiting the TGF-β1/Smad signaling pathway, thereby inhibiting the proliferation of myocardial fibroblasts, reducing collagen synthesis, and preventing the transformation of fibroblasts into myofibroblasts (α-SMA downregulation) [1]
2. In addition to its anti-fibrotic effect, FT 011 also exerts anti-inflammatory and anti-apoptotic effects in ischemic myocardium by inhibiting the NF-κB signaling pathway and regulating the Bcl-2/Bax balance, thereby helping to improve cardiac function and reduce cardiac remodeling [1]
3. FT 011 showed good safety in preclinical studies, and no obvious acute or chronic toxicity was observed at therapeutic doses. Its ability to improve cardiac function and reduce fibrosis in rats with myocardial infarction suggests that FT 011 has potential application value in treating cardiac dysfunction and fibrosis caused by myocardial infarction [1]

C₂₀H₁₇NO₅

351.35

351.11

C, 68.37; H, 4.88; N, 3.99; O, 22.77

1001288-58-9

23648966

Light yellow to yellow solid powder

1.3±0.1 g/cm3

618.9±55.0 °C at 760 mmHg

328.1±31.5 °C

0.0±1.9 mmHg at 25°C

1.656

4.39

2

5

7

26

564

0

O(CC#C)C1=CC=C(/C=C/C(NC2C=CC=CC=2C(=O)O)=O)C=C1OC

UIWZIDIJCUEOMT-PKNBQFBNSA-N

InChI=1S/C20H17NO5/c1-3-12-26-17-10-8-14(13-18(17)25-2)9-11-19(22)21-16-7-5-4-6-15(16)20(23)24/h1,4-11,13H,12H2,2H3,(H,21,22)(H,23,24)/b11-9+

2-[[(E)-3-(3-methoxy-4-prop-2-ynoxyphenyl)prop-2-enoyl]amino]benzoic acid

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 (7.12 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 (7.12 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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 (Please use freshly prepared in vivo formulations for optimal results.)