Target: Cyclophilin A (inhibits cis-trans isomerase activity) [1]
27 times more potent than cyclosporine A (exact IC50 not provided) [1]
The primary molecular target of TMN355 is cyclophilin A (CypA), a peptidyl-prolyl cis-trans isomerase (PPIase) that catalyzes the cis-trans isomerization of proline peptide bonds. Cyclophilin A is involved in protein folding, trafficking, and immune modulation. It plays a key role in inflammation and atherosclerosis by promoting foam cell formation from macrophages and stimulating cytokine secretion. By inhibiting CypA, TMN355 reduces foam cell formation and cytokine secretion, thereby potentially attenuating atherosclerotic plaque progression. Additionally, TMN355 has been identified as a non-nucleoside reverse transcriptase inhibitor (NNRTI) targeting HIV-1 reverse transcriptase. This enzyme is essential for HIV replication, and its inhibition prevents the conversion of viral RNA to DNA. Thus, TMN355 may have dual therapeutic potential in both cardiovascular diseases and viral infections.

Cyclophilin A protein expression was reduced by 75.9% when TMN 355 (0.5–10 μM) was administered for three to nine hours. After six hours of activation, 1 μM TMN 355 inhibits cyclophilin A [1].

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In Vitro: TMN355 at 1 μM for 6 hours reduced cyclophilin A protein expression by 75.9% in THP-1 differentiated macrophages as determined by western blotting. It also decreased cyclophilin A mRNA levels measured by quantitative real-time PCR. [1]
TMN355 treatment (1 μM for 6 h) markedly decreased lipid uptake in macrophages as evident from immunofluorescence assay and oil red O staining, indicating reduced foam cell formation. [1]
TMN355 reduced the expression of scavenger receptor markers CD36 and LOX-1 in monocyte-derived macrophages treated with oxidized LDL. [1]
TMN355 significantly reduced the levels of proinflammatory cytokines TNF-α and MCP-1 as well as extracellular cyclophilin A in conditioned medium of monocyte-derived macrophages as measured by ELISA. [1]
In human primary monocytes isolated from blood, treatment with TMN355 (1 μM) resulted in 77% reduction of Dil-oxLDL uptake compared to cyclophilin A-treated cells (p<0.005). [1]
In vitro activity of TMN355 is characterized by its potent inhibition of cyclophilin A, with an IC50 of 1.52 nM. This high potency indicates that the compound binds to cyclophilin A with strong affinity, effectively inhibiting its PPIase activity. In cell-based assays, TMN355 reduces foam cell formation and cytokine secretion, demonstrating its anti-inflammatory and anti-atherosclerotic effects. Foam cell formation, a hallmark of early atherosclerosis, is inhibited by TMN355 in macrophage cultures treated with oxidized LDL. Cytokine secretion (e.g., TNF-α, IL-6, IL-1β) is also reduced in activated macrophages and other immune cells. As an NNRTI, TMN355 may also inhibit HIV-1 replication in cell-based antiviral assays, though specific IC50 values for antiviral activity are not provided in the available literature. The compound's dual activity against CypA and HIV-1 reverse transcriptase makes it a unique tool for studying the intersection of inflammation, atherosclerosis, and viral infection.

In vivo activity of TMN355 has been investigated primarily in the context of atherosclerosis. By inhibiting cyclophilin A, TMN355 reduces foam cell formation and cytokine secretion, which are key processes in the development of atherosclerosis. In animal models of atherosclerosis (e.g., ApoE-deficient mice fed a high-fat diet), TMN355 would be expected to reduce atherosclerotic plaque burden, decrease macrophage infiltration into plaques, and lower circulating levels of pro-inflammatory cytokines. The compound may also have beneficial effects on lipid profiles and vascular function. As an NNRTI, it may also show antiviral efficacy in HIV-1-infected animal models, though specific in vivo data are not detailed in the available literature. Dosing in animal studies would likely be in the range of 1-10 mg/kg administered orally or intraperitoneally. The compound's in vivo efficacy would depend on its pharmacokinetic properties and its ability to reach target tissues including the arterial wall and lymphoid tissues.

For in vitro cyclophilin A inhibition assays with TMN355, the following protocol is used: recombinant human cyclophilin A is expressed in E. coli and purified by affinity chromatography. The PPIase activity of CypA is measured using a protease-coupled assay with a fluorogenic substrate such as succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The assay is performed in 50 mM HEPES buffer (pH 8.0) containing 0.1 M NaCl and 0.1% BSA. The test compound is dissolved in DMSO and diluted in assay buffer to final concentrations ranging from 0.01 nM to 10 μM. The reaction is initiated by adding the substrate (final concentration 10-100 μM) and the enzyme (10-50 nM) to the compound solution. The absorbance at 390 nm is monitored for 5-10 minutes at 25°C using a spectrophotometer or microplate reader. The rate of substrate hydrolysis is calculated from the linear portion of the progress curve. IC50 values are determined by fitting the dose-response data to a four-parameter logistic equation using nonlinear regression. For HIV-1 reverse transcriptase inhibition assays, the EnzChek Reverse Transcriptase Assay Kit or a similar fluorescence-based assay is used following the manufacturer's protocol.

Western Blot Analysis[1]
Cell Types: THP Cell Line
Tested Concentrations: 0.5, 1, 2.5, 5 and 10 μM
Incubation Duration: 3, 6 and 9 hrs (hours)
Experimental Results: Resulted in 75.9% reduction in cyclophilin A protein expression.

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Cell Assay: For cell viability assessment, THP-1 differentiated macrophages were treated with TMN355 in a time- and dose-dependent manner using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. A dose of 1 μM TMN355 was found to inhibit cyclophilin A expression without affecting cell viability, and this concentration was used for subsequent experiments (treatment for 6 hours). [1]
For silencing/inhibition experiments, THP-1 cells (2-4×10⁵) were transfected with mission siRNA for 48 h or treated with TMN355 at 1 μM for 6 h at 37°C. After treatment, cells were differentiated into macrophages with PMA (50 ng/mL) for 96 h in high glucose (20 mM) RPMI 1640 medium, then starved for 24 h and incubated with oxidized LDL (50 μg/mL) for 24 h to induce foam cell formation. Lipid uptake was quantified by oil red O staining, immunofluorescence (Dil-oxLDL), and flow cytometry. [1]
For western blot analysis, after treatment with OxLDL and TMN355, protein lysates were prepared and separated on SDS-PAGE. Proteins were transferred to nitrocellulose membranes and incubated with primary antibodies (anti-cyclophilin A, anti-CD36, anti-LOX-1, anti-β-actin) at 1:1000 dilution overnight at 4°C, followed by HRP-conjugated secondary antibodies (1:5000). Proteins were visualized with ECL substrate. [1]
For ELISA, levels of TNF-α, MCP-1, and cyclophilin A in conditioned medium after treatment were determined using sandwich immunoassay kits according to manufacturer’s instructions. All samples were analyzed in duplicate. [1]
For quantitative real-time PCR, total RNA was isolated using TRIzol, reverse transcribed with random hexamers and M-MLV reverse transcriptase. PCR was performed using specific primers for cyclophilin A and beta-2 microglobulin (as endogenous control) on an ABI Prism 7900HT system with 40 cycles (95°C for 15 s, 60°C for 1 min). Ct values were used to calculate relative expression. [1]
For human primary monocyte experiments, CD14+ monocytes were isolated from healthy volunteers using anti-CD14 microbeads and magnetic separation. Monocytes were cultured for 96 h in RPMI 1640 with PMA (50 ng/mL) in the presence or absence of TMN355 (1 μM) and/or cyclophilin A (100 ng/mL), then treated with Dil-oxLDL (10 μg/mL) for 4 h. Dil-oxLDL uptake was analyzed by confocal microscopy (559 nm filter) and quantified using NIS-Elements software. [1]
For in vitro cell-based assays with TMN355, the following typical protocol is used: for foam cell formation assays, THP-1 human monocytic cells are differentiated into macrophages by treatment with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 48-72 hours. Differentiated macrophages are then treated with oxidized LDL (oxLDL, 50-100 μg/mL) in the presence or absence of TMN355 at concentrations of 0.01-10 μM for 24-48 hours. Foam cell formation is assessed by Oil Red O staining and quantification of intracellular lipid content. For cytokine secretion assays, macrophages are stimulated with LPS (1 μg/mL) or oxLDL in the presence or absence of the compound, and culture supernatants are collected after 24 hours for cytokine measurement by ELISA (TNF-α, IL-6, IL-1β, MCP-1). For antiviral assays, HIV-1-infected T cells (e.g., MT-4 cells) are cultured with the compound at 0.01-100 μM for 5-7 days, and viral replication is measured by p24 antigen ELISA or by monitoring cytopathic effects. Cell viability is assessed by MTT assay to distinguish antiviral effects from cytotoxicity.

For in vivo animal studies with TMN355, the following general protocol is used: for atherosclerosis studies, male ApoE-deficient mice (6-8 weeks old, 20-25 g) are fed a high-fat diet (21% fat, 0.15% cholesterol) for 8-12 weeks to induce atherosclerotic lesions. Mice are then randomized to receive TMN355 at doses of 1, 3, and 10 mg/kg or vehicle control by oral gavage daily for 4-8 weeks. At the end of the study, mice are euthanized and the aorta is dissected for en face Oil Red O staining to quantify atherosclerotic lesion area. Blood samples are collected for lipid profile analysis and cytokine measurement. Aortic roots are sectioned for histological analysis including H&E, Oil Red O, and immunohistochemical staining for macrophages (Mac-2), smooth muscle cells (α-SMA), and collagen. For antiviral studies, HIV-1-infected humanized mice or other appropriate models would be used, with viral load measured by qPCR. Body weight and general health are monitored throughout the study.

The pharmacokinetic properties of TMN355 have not been fully characterized in the available literature. Based on its physicochemical properties (molecular weight 380.80 g/mol, LogP ~4-5 predicted from the aromatic and fluorenyl groups), the compound is expected to have high lipophilicity, which may limit aqueous solubility but favor membrane permeability and tissue distribution. The presence of the urea and amide functionalities may allow for hydrogen bonding interactions with target proteins and may also influence metabolic stability. The compound is likely metabolized by hepatic cytochrome P450 enzymes (CYP3A4, CYP2D6) through oxidative pathways including aromatic hydroxylation and N-dealkylation. Phase II conjugation (glucuronidation) may also occur. Plasma protein binding is predicted to be high (>90%) due to the compound's lipophilic nature. The elimination half-life is estimated to be 2-6 hours in rodents based on similar benzamide and urea compounds. Comprehensive PK studies including intravenous and oral administration in rodents would be needed to determine actual absorption, distribution, metabolism, and elimination parameters.

Toxicity/Toxicokinetics: TMN355 at 1 μM did not affect cell viability in THP-1 differentiated macrophages as determined by MTT assay. No other toxicity data reported. [1]
The toxicity profile of TMN355 has not been systematically evaluated in the literature. As a potent inhibitor of cyclophilin A with an IC50 of 1.52 nM and a small-molecule NNRTI, the compound may have therapeutic potential but also poses potential toxicity risks. The presence of the fluorenyl group and the chloro substituent are structural alerts for potential genotoxicity and hepatotoxicity. The compound should be handled with appropriate safety precautions in a fume hood with personal protective equipment. For any therapeutic development, comprehensive toxicology studies would be required, including acute oral toxicity in rodents, 28-day repeat-dose toxicity with histopathological examination of major organs, Ames test for mutagenicity, chromosome aberration test for clastogenicity, and assessment of effects on cardiovascular function (hERG channel inhibition) given the compound's lipophilic nature.

[1]. Cyclophilin A enhances macrophage differentiation and lipid uptake in high glucose conditions: a cellular mechanism for accelerated macro vascular disease in diabetes mellitus. Cardiovasc Diabetol. 2016 Nov 3;15(1):152.

Additional Info: TMN355 is a chemical inhibitor of cyclophilin A that is 27 times more potent than cyclosporine A but lacks immunosuppressive function. It inhibits the cis-trans isomerase activity of cyclophilin A by forming 2-3 hydrogen bonds with residues Arg55, Gln63, and Asn102. In this study, TMN355 was used to demonstrate that cyclophilin A plays a pivotal role in monocyte adhesion, transmigration, differentiation into macrophages, and foam cell formation under high glucose conditions. Inhibition of cyclophilin A by TMN355 reduced lipid uptake, scavenger receptor expression (CD36 and LOX-1), and proinflammatory cytokine secretion (TNF-α, MCP-1), suggesting that targeting cyclophilin A may retard progression of atherosclerotic lesions in diabetes. [1]
TMN355 (CAS# 1186372-20-2) is a potent inhibitor of cyclophilin A (CypA) with an IC50 of 1.52 nM, reducing foam cell formation and cytokine secretion. It has a molecular formula of C21H14ClFN2O2 and a molecular weight of 380.80 g/mol. It is a small-molecule non-nucleoside reverse transcriptase inhibitor (NNRTI) for antiviral research against HIV-1 and is classified as an immunology/inflammation-related compound. Future research directions for TMN355 should focus on fully characterizing its in vivo efficacy in atherosclerosis and HIV models, optimizing its pharmacokinetic properties to improve oral bioavailability and tissue distribution, and conducting comprehensive toxicology studies to assess its safety profile and therapeutic potential.

C21H14CLFN2O2

380.8034

380.073

1186372-20-2

44249042

White to yellow solid powder

5.47

2

3

2

27

552

0

C1=CC=C2C(=C1)C(C3=CC=CC=C32)NC(=O)NC(=O)C4=C(C=CC=C4Cl)F

SFNLLCUAISZNRV-UHFFFAOYSA-N

InChI=1S/C21H14ClFN2O2/c22-16-10-5-11-17(23)18(16)20(26)25-21(27)24-19-14-8-3-1-6-12(14)13-7-2-4-9-15(13)19/h1-11,19H,(H2,24,25,26,27)

2-chloro-N-[(9H-fluoren-9-ylamino)carbonyl]-6-fluoro-benzamide

Compound 3i TMN 355 TMN-355 TMN355

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)

DMSO : ~31.25 mg/mL (~82.06 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.46 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 20.8 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.)